The application of electrical engineering principles to biology represents the main issue of bioelectronics, focusing on interfacing of electronics with biological systems. In particular, it includes many applications that take advantage of the peculiar optoelectronic and mechanical properties of organic or inorganic semiconductors, from sensing of biomolecules to functional substrates for cellular growth. Among these, technologies for interacting with bioelectrical signals in living systems exploiting the electrical field of biomedical devices have attracted considerable attention. In this review, we present an overview of principal applications of phototransduction for the stimulation of electrogenic and non-electrogenic cells focusing on photovoltaic-based platforms.

Photocurrent therapy with participation of light and electrical stimulations could be an innovative and promising approach in regenerative medicine, especially for skin and nerve regeneration. Photocurrent is generated when light irradiates on a photosensitive device, and with more and more types of photosensitive materials being synthesized, photocurrent could be applied for enhanced regeneration of tissue. Photosensitive scaffolds such as composite poly (3-hexylthiophene)/polycaprolactone (P3HT/PCL) nanofibers are fabricated by electrospinning process in our lab for skin regeneration in presence of applied photocurrent. This review article discuss on the various in vitro, in vivo and clinical studies that utilized the principle of 'electrotherapy' and 'phototherapy' for regenerative medicine and evaluates the potential application of photocurrent in regenerative medicine. We conclude that photocurrent therapy will play an important role in regenerative medicine.

The guinea pig spermatozoid nucleus contains actin, myosin, spectrin and cytokeratin. Also, it has been reported that phalloidin and/or 2,3-butanedione monoxime retard the sperm nuclear decondensation caused by heparin, suggesting a role for F-actin and myosin in nuclear stability. The presence of an F-actin/myosin dynamic system in these nuclei led us to search for proteins usually related to this system. In guinea pig sperm nuclei we detected calmodulin, F-actin, the myosin light chain and an actin-myosin complex. To define whether calmodulin participates in nuclear-dynamics, the effect of the calmodulin antagonists W5, W7 and calmidazolium was tested on the decondensation of nuclei promoted by either heparin or by a Xenopus laevis egg extract. All antagonists inhibited both the heparin- and the X. laevis egg extract-mediated nuclear decondensation. Heparin-mediated decondensation was faster and led to loss of nuclei. The X. laevis egg extract-promoted decondensation was slower and did not result in loss of the decondensed nuclei. It is suggested that in guinea pig sperm calmodulin participates in the nuclear decondensation process.

Using a continuous superfusion system of dog renal cortical slices, we studied the role of Ca(2+) in the intracellular control mechanism for renin release. The calcium ionophore A23187 (10 microM) produced a significant decrease in renin release. This effect was abolished in the absence of extracellular Ca(2+). Moreover, pretreatment with the calmodulin inhibitor W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, 20 microM) completely prevented the inhibitory effect of A23187 (10 microM). The beta-adrenoceptor agonist isoproterenol (1, 10 and 100 microM) produced a concentration-dependent increase in renin release. Pretreatment with W-7 (20 microM) potentiated the stimulation of renin release induced by isoproterenol (1 microM). These results suggest that A23187-induced inhibition of renin release is mediated by the activation of calmodulin via an increase in intracellular Ca(2+) and beta-adrenoceptor-stimulated renin release is modulated by intracellular Ca(2+) mobilization.

Electrical stimulation is used to treat nonunions and to augment spinal fusions. We studied the biochemical pathways that are activated in signal transduction when various types of electrical stimulation are applied to bone cells.

Cultured MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields at appropriate field strengths for thirty minutes and for two, six, and twenty-four hours. The DNA content of each dish was determined. Other cultures of MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields for two hours in the presence of various inhibitors of signal transduction, with or without electrical stimulation, and the DNA content of each dish was determined.

All three signals produced a significant increase in DNA content per dish compared with that in the controls at all time-points (p < 0.05), but only exposure to capacitive coupling resulted in a significant, ever-increasing DNA production at each time-period beyond thirty minutes. The use of specific metabolic inhibitors indicated that, with capacitive coupling, signal transduction was by means of influx of Ca(2+) through voltage-gated calcium channels leading to an increase in cytosolic Ca(2+) (blocked by verapamil), cytoskeletal calmodulin (blocked by W-7), and prostaglandin E2 (blocked by indomethacin). With inductive coupling and combined electromagnetic fields, signal transduction was by means of intracellular release of Ca(2+) leading to an increase in cytosolic Ca(2+) (blocked by TMB-8) and an increase in activated cytoskeletal calmodulin (blocked by W-7).

The initial events in signal transduction were found to be different when capacitive coupling was compared with inductive coupling and with combined electromagnetic fields; the initial event with capacitive coupling is Ca(2+) ion translocation through cell-membrane voltage-gated calcium channels, whereas the initial event with inductive coupling and with combined electromagnetic fields is the release of Ca(2+) from intracellular stores. The final pathway, however, is the same for all three signals-that is, there is an increase in cytosolic Ca(2+) and an increase in activated cytoskeletal calmodulin.

Several antipsychotic drugs reverse the dopamine-induced inhibition of prolactin release by rat pituitary cell cultures. Paradoxically, at high doses and without dopamine, antipsychotic drugs can also inhibit prolactin secretion. The mechanism underlying this phenomenon is unclear. Some evidence suggests that these drugs have an agonistic action. We sought to verify whether clozapine and fluphenazine, at doses higher than those reversing dopamine-induced inhibition of prolactin secretion in vitro, show this paradoxical effect and eventually a partial agonistic action. Both antipsychotics inhibited prolactin secretion, clozapine at doses starting from 10(-6) M and fluphenazine from 10(-7) M. Haloperidol reversed clozapine-induced prolactin inhibition but left fluphenazine-induced inhibition unchanged. These in vitro findings suggest that clozapine has a partial agonistic action on dopaminergic receptors but fluphenazine does not.

Invasion through stromal extracellular matrix (ECM) is part of the complex, multistep process of tumour cell invasion and metastasis. Our group has previously demonstrated that calcium and calmodulin are important in another step in the metastatic cascade - that of attachment of cells to ECM. Interestingly, the non-steroidal anti-oestrogen tamoxifen (which also has calmodulin antagonist activity), used in the treatment of breast cancer and now in metastatic cutaneous melanoma, can inhibit the attachment of normal and neoplastic cells to ECM. In this study, we investigated whether such drugs, known to inhibit cell attachment, could also subsequently reduce their invasion through a layer of human fibronectin. We examined the ability of the specific calmodulin antagonist J8, tamoxifen and its two major metabolites, N-desmethyltamoxifen (N-des) and 4-hydroxytamoxifen (4-OH), as well as the pure anti-oestrogen ICI 182,780 and 17beta-oestradiol to inhibit invasion of the human cutaneous melanoma cell line, A375-SM, uveal melanoma cells and uveal melanocytes. A375-SM cells and uveal melanoma cells showed a high level of invasion (15.2% and 33.7% respectively) compared with melanocytes (around 5%) under the experimental conditions used. Submicromolar concentrations of N-des, tamoxifen, J8 and 17beta-oestradiol significantly reduced the invasiveness of the A375-SM cell line. The uveal melanoma cells also showed similar inhibition, although at higher concentrations of these agents. 4-OH and ICI 182, 780 had little or no effect on invasion of A375-SM cells (these were not tested on uveal melanoma cells). All cells used in this study were found to be negative for type I nuclear oestrogen receptors, reinforcing the possibility that tamoxifen and 17beta-oestradiol can act via mechanisms unrelated to binding to classical oestrogen receptors to inhibit tumour cell invasion.

MK-886, a leukotriene biosynthesis inhibitor, which prevents the translocation and activation of 5-lipoxygenase, has been proposed as an effective drug for the treatment of inflammatory disorders, including psoriasis. In the present study, we investigated the effects of MK-886 on calmodulin as a potential target protein of anti-inflammatory drug activity, and on the proliferation of cultured human keratinocytes, a calmodulin-dependent cellular response with indicative value for antipsoriatic drug activity. Despite potent calmodulin-antagonistic activity in vitro, MK-886 failed to block cell proliferation in a human keratinocyte line, whereas trifluoperazine, a well characterized calmodulin antagonist with similar effects on calmodulin activity in our in vitro assays, inhibited cell proliferation in a dose-dependent manner. Further investigations on the mechanism of action revealed that, in contrast with trifluoperazine, calmodulin antagonism by MK-886 in vitro is likely to be mediated at the level of the allosteric calmodulin-recognition site of phosphodiesterase, rather than by binding to calmodulin itself. Therefore, our data do not conflict with the proposed role of calmodulin in the regulation of cell proliferation, but demonstrate that drug-induced antagonism of calmodulin, detected by inhibition of calmodulin-dependent enzymes in vitro, is not necessarily linked to antiproliferative activity in human keratinocytes.

Transgenic mice carrying a fused gene of the 294-base upstream and 68-base leader sequences of a rat calmodulin gene, CaMII, and beta-galactosidase gene were made. Only spermatocytes expressed the transgene mRNA in the testes of four independent transgenic lines. The localization of transgene mRNA was consistent with that of the mouse endogenous CaMII analyzed by in situ hybridization with the probe of 3'-noncoding region of mouse CaMII. Thus, this short promoter of CaMII evidently conferred the expression of transgene only on spermatocytes but not on spermatogonia nor on spermatids of the testis. The rat CaMII promoter up to -294 contained no sequences that corresponded to any of the reported sequence features of genes expressed in the testis. Therefore, this short promoter region of CaMII seemed to carry a certain novel machinery for the spermatocyte-specific gene expression.

Pneumocystis carinii pneumonia (PCP) is a leading cause of death among AIDS patients in the United States. Our analysis of P. carinii protein-coding genes has revealed a significant A + T codon bias. Polymerase chain reaction (PCR) was utilized to isolate and identify the genes encoding calmodulin, beta-tubulin, DNA polymerase II, and RNA polymerases I, II and III from P. carinii. Primer pairs were designed to incorporate P. carinii codon preference to known conserved protein regions from other organisms. This strategy should be useful for a large variety of P. carinii genes and assist in the comprehensive analysis of the genomic structure of this important pathogen.

Azole derivatives, such as ketoconazole and bifonazole, are well-established antifungal drugs. Recently, these compounds have been reported to have therapeutic efficacy also in inflammatory skin disorders. There is increasing evidence that calmodulin is involved in fungal infections as well as in inflammatory skin diseases. Therefore, we investigated the effects of various antifungal drugs on calmodulin activity, using calmodulin-dependent phosphodiesterase as an indicator for the calmodulin activity. All azole derivatives tested competitively inhibited calmodulin activity with 50% inhibitory concentration values in the low micromolar range. In contrast, antifungal drugs belonging to other chemical classes did not display inhibitory activity. Thus, this study provides evidence that direct interaction with calmodulin might contribute to the therapeutic activity of azole derivatives, particularly to their efficacy in the treatment of inflammatory skin disorders.

Normal human umbilical vein endothelial cells cultured on gelatin-coated plastic dishes were found to produce a protein in their media which had calmodulin-like immunoreactivity and biological activity. Further identification of the protein was achieved by examining the incorporation of 14C leucine into protein found in the conditioned medium. Cells produced 14C labelled protein in their medium which specifically bound to an affinity column for calmodulin. This latter material stimulated calmodulin dependent phosphodiesterase activity in vitro and this stimulation was inhibited by the addition of the calmodulin antagonist W7. The presence of calmodulin-like activity and immunoreactivity in the media varied as the cells grew from low to high density, a peak of extracellular calmodulin-like activity preceding an increase in cell number. Extracellular calmodulin-like activity did not correlate with the presence of lactate dehydrogenase in the medium. The addition of pure pig brain calmodulin affected the rate of cell proliferation; significant proliferation to pure calmodulin was only seen in cells at low density, at higher density calmodulin either had no effect or inhibited proliferation. Inhibition of extracellular calmodulin activity by a calmodulin antagonist immobilized on agarose beads, or by an antibody to calmodulin significantly decreased proliferation in all dividing cultures. Taken together this data suggests that, in vitro, calmodulin, or a very closely related protein, influences endothelial cell proliferation through an autocrine mechanism.

Isolated newborn rat calvarial bone cells grown in monolayer on polyurethane membranes in specially constructed culture chambers and subjected to a cyclical biaxial mechanical strain of 0.17% at a frequency of 1 Hz for 30 min demonstrated a 16% increase in DNA synthesis during the subsequent 24 h. The metabolites of the inositol phosphate pathway, shown to be an important second messenger in many cell types, were shown to be elevated using high-performance liquid chromatography to separate and quantitate the various inositol polyphosphates. Inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1,3,4,5-tetrakisphosphate reached peak accumulations after 20 s of mechanical strain. Inositol 1,3,4-trisphosphate reached a peak accumulation after 2 min, and inositol 1,2,3,4,5,6 phosphate reached a peak accumulation after 60 min of mechanical strain. Neomycin, an inhibitor of phospholipase C, a membrane-bound enzyme that hydrolyzes phosphatidyl inositol 4,5-bisphosphate to start the inositol phosphate cascade, completely inhibited accumulation of the above inositol phosphates during mechanical straining of the bone cells. Neomycin also completely abolished the increase in DNA synthesis that was seen after a mechanical strain of 0.17%. It is concluded from this study that the inositol phosphate pathway is activated by mechanical strain in bone cells and that this pathway is an important and primary mediator in the transduction of mechanical strain into cellular proliferation in these cells.

Calmodulin is an important modulator of intracellular calcium processes and may be implicated in the calcium malabsorption of coeliac disease. The calmodulin content in extracts of duodenal biopsy specimens from 48 normal control subjects and 28 patients with coeliac disease was determined. Radioimmunoassay was used to measure immunoreactive calmodulin while a cyclic adenosine 3',5'-monophosphate phosphodiesterase activity assay was used to measure biologically active calmodulin. Calmodulin values measured by both assays were similar for control and disease groups. Mean (SEM) immunoreactive calmodulin values were 1.68 (0.09) micrograms/mg protein for controls and 1.67 (0.15) and 1.45 (0.15) micrograms/mg protein for partial and total villous atrophy respectively. These values were not significantly different. Biologically active calmodulin values were 2.77 (0.21), 1.82 (0.34), and 3.24 (0.33) micrograms/mg protein for control, partial, and total villous atrophy subjects respectively. The biologically active calmodulin values in the partial villous atrophy group were significantly lower than in controls and total villous atrophy subjects. In the phosphodiesterase assay, the calmodulin antagonist trifluoperazine inhibited the activity stimulated by purified calmodulin and by the extracts to the same extent. These results show that calmodulin values are normal in coeliac disease and provide no evidence that changes in calmodulin account for the abnormal calcium absorption in these patients.

The calmodulin and calcium dependence of human adenylate cyclase from the second part of the duodenum was assessed in washed particulate preparations of biopsy specimens by investigating (a) the concentration dependent effects of free [Ca2+] on enzyme activity, (b) the effects of exogenous calmodulin on enzyme activity in ethylene glycol bis (b-aminoethyl ether)N,N'-tetra-acetic acid (EGTA) washed particulate preparations, and (c) the effects of calmodulin antagonists on enzyme activity. Both basal (IC50 = 193.75 (57.5) nmol/l (mean (SEM)) and NaF stimulated (IC50 = 188.0 (44.0) nmol/l) adenylate cyclase activity was strongly inhibited by free [Ca2+] greater than 90 nmol/l. Free [Ca2+] less than 90 nmol/l had no effect on adenylate cyclase activity. NaF stimulated adenylate cyclase activity was inhibited by 50% at 2.5 mmol/l EGTA. This inhibition could not be reversed by free Ca2+. The addition of exogenous calmodulin to EGTA (5 mmol/l) washed particulate preparations failed to stimulate adenylate cyclase activity. Trifluoperazine and N-(8-aminohexyl)-5-IODO-1-naphthalene-sulphonamide (IODO 8) did not significantly inhibit basal and NaF stimulated adenylate cyclase activity when measured at concentrations of up to 100 mumol/l. These results suggest that human duodenal adenylate cyclase activity is calmodulin independent but is affected by changes in free [Ca2+].

Various studies have suggested that calmodulin (CaM) is involved in the pathophysiology of psoriasis. Protein kinase C (PKC) is also accepted as playing a regulatory role in cell proliferation as well as in inflammatory processes. Therefore, we investigated the effects of the known CaM antagonist tiflucarbine (BAY/TVX P 4495) on two cellular systems related to the major clinical symptoms of psoriasis: proliferation of cultured human keratinocytes (HaCa T cell line) and release of reactive oxygen species (ROS) from human polymorphonuclear leukocytes (PMNL). Tiflucarbine inhibited both cellular responses in a dose dependent manner. Furthermore, tiflucarbine directly affected PKC, and may thus be considered to be a dual PKC/CaM antagonist with putative antipsoriatic activity. The effects of tiflucarbine on the different parameters were compared with those of the structurally unrelated dual PKC/CaM inhibitor W-7 and those of the potent PKC inhibitor staurosporine. The potencies of all three compounds were found to be in the same range as their PKC-inhibiting potency. Our data indicate that PKC, rather than CaM, may play a regulatory role in the release of ROS as well as in keratinocyte proliferation. Therefore, inhibition of PKC in general might have a therapeutic benefit in psoriasis.

We examined calcium and calmodulin regulation of atrial natriuretic factor stimulation of particulate-membrane guanylate cyclase (ANF-s-GC) in SK-NEP-1 cells. W7 and trifluoropiperazine, but not W5, inhibited whole cellular ANF-stimulated cyclic GMP accumulation (ANF-s-cGMP). EGTA and LaCl3 decreased ANF-s-GC and calmodulin reversed this inhibition. A23187-induced inhibition of ANF-s-cGMP was only partly reversible by IBMX. H7 or staurosporine counteracted the inhibitory effect of A23187. Calcium inhibited basal and ANF-s-GC. These data suggest that at low concentrations of calcium, ANF-s-GC was calcium-calmodulin dependent but high concentrations of calcium inhibited ANF-s-GC through phosphodiesterase, through inhibition of GC, and probably through protein kinase C.

Polymyxin B, a cyclic peptide antibiotic, is considered to be a rather selective antagonist of protein kinase C. This drug is therefore widely used to evaluate the involvement of protein kinase C in cellular processes. In the present study, we investigated the effects of polymyxin B on the activity of calmodulin-dependent cyclic 3':5'-nucleotide phosphodiesterase in vitro. The drug potently inhibited this enzyme (IC50 80 nM in the presence of 500 microM Ca2+), while about 200-fold higher concentrations were required to inhibit protein kinase C to the same extent. Phosphodiesterase inhibition was competitive with respect to Ca2+ and calmodulin. Evidence for the formation of a complex between polymyxin B and calmodulin was obtained by polyacrylamide gel electrophoresis under non-denaturing conditions, and by affinity chromatography of calmodulin on polymyxin B-agarose. We therefore suggest that, at least in vitro, polymyxin B is a potent and selective inhibitor of calmodulin.

Clozapine is an antipsychotic drug which is unusual in that it has no dopamine receptor-blocking activity. Previous studies gave conflicting results whether administration of clozapine induces hyperprolactinemia. In the present study it was shown that a wide concentration range of clozapine does not interfere with dopamine-mediated inhibition of prolactin (PRL) secretion by normal cultured rat pituitary cells. This in contrast to other neuroleptics, like haloperidol and trifluoperazine. Clozapine does also not antagonize norepinephrine-mediated inhibition of PRL secretion. Clozapine exerts at micromolar concentrations a direct inhibitory action on PRL release by cultured normal rat pituitary cells. In cultured rat pituitary tumor cells, these high concentrations of clozapine directly inhibit PRL release as well as the DNA content of the cells, suggesting a direct antimitotic action. In this model clozapine was about 5-10 times less potent than trifluperazine. Clozapine and trifluoperazine exert an additive inhibitory action both on PRL release and on the DNA content of the pituitary tumor cells. It is concluded that clozapine does not interfere at the pituitary level with dopamine-mediated inhibition of PRL release. At micromolar concentrations clozapine may act on lactotrophs as a calmodulin-inhibitor. These observations suggest that the transient PRL-releasing effects which have been observed in both animal and human studies after clozapine administration are mediated via supra-pituitary actions of the drug.

Vitamin D3 and its active metabolite I alpha-25-dihydroxyvitamin D3 (I alpha-25-(OH)2D3) have been reported to play a role in melanogenesis. Physiological concentrations of I alpha-25-(OH)2D3 were found to acutely elevate intracellular free calcium (using Fura 2) in B16 primary (Io) cells. Membrane phosphoinositide turnover was unaffected by I alpha-25-(OH)2D3. The rise in intracellular free calcium was entirely dependent on extracellular calcium and was not mimicked by vitamin D3. However, in neither B16-Io nor B16-F1 melanoma cells did vitamin D3 or I alpha-25-(OH)2D3 increase melanin production.

Calmodulin-binding proteins present in chromaffin cell plasma membranes were isolated and directly compared with calmodulin-binding proteins present in chromaffin granule membranes. Chromaffin cell plasma membranes were prepared using Cytodex 1 microcarriers. Marker enzyme studies on this preparation showed a nine- to 10-fold plasma membrane enrichment over cell homogenates and a low contamination of these plasma membranes by subcellular organelles. Plasma membranes prepared in this manner were solubilized with Triton X-100 and applied to a calmodulin-affinity column in the presence of calcium. Several major calmodulin-binding proteins (240, 105, and 65 kilodaltons) were eluted by an EGTA-containing buffer. 125I-Calmodulin overlay experiments on nitrocellulose sheets containing both chromaffin plasma and granule membranes showed that these two membranes have several calmodulin-binding proteins in common (65, 60, 53, and 50 kilodaltons), as well as unique calmodulin-binding proteins (34 kilodaltons in granule membranes and 240 and 160 kilodaltons in plasma membranes). The 65-kilodalton calmodulin-binding protein present in both membrane types was shown to consist of two isoforms (pI 6.0 and 6.2) by two-dimensional gel electrophoresis. Previous experiments from our laboratory, using two monoclonal antibodies (mAb 30 and mAb 48) specific for a rat brain synaptic vesicle membrane protein (p65), showed that the monoclonal antibodies reacted with a 65-kilodalton calmodulin-binding protein present in at least three neurosecretory vesicles (chromaffin granules, neurohypophyseal granules, and rat brain synaptic vesicles). When these monoclonal antibodies were tested on chromaffin cell plasma membranes and calmodulin-binding proteins isolated from these membranes, they recognized a 65-kilodalton protein. These results indicate that an immunologically identical calmodulin-binding protein is expressed in both chromaffin granule membranes (as well as other secretory vesicle membranes) and chromaffin cell plasma membranes, thus suggesting a possible role for this protein in granule/plasma membrane interaction.

1. Addition of extracellular pure pig brain calmodulin was found to modulate DNA synthesis and cell proliferation in K562 human leukaemic lymphocytes. At lower cell densities calmodulin significantly stimulated [3H]thymidine uptake; at higher densities it decreased it. 2. A protein biochemically indistinguishable from calmodulin was detected in the cell-conditioned media of rapidly dividing K562 cells. The concentration of calmodulin-like activity found in the conditioned media of these and a range of other normal and neoplastic cells (250-1636 ng/ml) was of the same order as would stimulate DNA synthesis in subconfluent cells. 3. Amounts of extracellular calmodulin-like activity and immunoreactivity varied during cell growth from low to high density, a peak of extracellular calmodulin preceding DNA synthesis in synchronized K562 cells. Extracellular calmodulin concentrations did not correlate with the presence of lactate dehydrogenase in the medium. 4. Inhibition of extracellular calmodulin activity by calmodulin antagonist immobilized on agarose beads, or by antibody to calmodulin, significantly decreased DNA synthesis. 5. These data strongly suggest that calmodulin or a very closely related protein can influence mitosis through an extracellular mechanism.

1. Effects of calcium-interacting agents on the calcium-induced stimulation of renin release from kidney cortical slices pretreated with calcium-free medium were examined. 2. The exposure of calcium to the slices pretreated with calcium-free medium enhanced the release of renin, followed by a decreased response in the release. The amount of lactate dehydrogenase released from the slices did not correlate with that of renin. 3. High potassium depolarization significantly potentiated the decreased response of renin release, with no influence on the stimulation of the release by exposure to calcium. 4. The decrease in renin release was attenuated by calcium-interacting agents, such as nifedipine, TMB-8 and W-7, but these agents were without effect on the stimulation of the release by exposure to calcium. 5. Thus, the calcium-calmodulin system apparently is not involved in the increased response of renin release following exposure to calcium.

In membranes prepared from the rat cerebral cortex EGTA 0.4-100 mumol/l was found to dose-dependently inhibit adenylate cyclase activity, both during basal conditions and when the cyclase activity had been stimulated with 10 mumol/l forskolin. Addition of calcium 2-30 mumol/l (in excess of EGTA) totally prevented the inhibition induced by EGTA, both in the absence and presence of forskolin. These data are consistent with the notion that in the absence of EGTA the rat cortex adenylate cyclase is considerably stimulated by endogenous calcium-calmodulin. The data also show that stimulation with endogenous calcium-calmodulin is more than additive with that of forskolin indicating an action on identical cyclase units. The kinetics of the rat cortex adenylate cyclase for its substrate (MgATP) was also investigated. Activation of the cyclase with endogenous calcium-calmodulin, induced a marked increase in the Vmax of the enzyme and a concomitant almost 2-fold increase in the Km. Stimulation with 10 microM forskolin also induced a marked increase in the Vmax as well as an almost 3-fold increase in the Km. A combination of the two stimulants caused a further increase in Vmax as well as Km; the Vmax being increased more than 17-fold and the Km being increased 3.5-fold over the basal. The data indicates that both calcium-calmodulin and forskolin induces a similar type of activation of the adenylate cyclase which is associated with a lowered affinity for its substrate.

Calmodulin content was evaluated in 3 prolactin-secreting pituitary adenomas (prolactinoma) and 3 normal anterior pituitary glands. The calmodulin content in the normal anterior pituitary tissue was quite consistent, 3.32 +/- 0.016 micrograms/mg protein. In contrast, calmodulin content varied almost 4-fold in the prolactinoma tissue (10.97, 8.50 and 3.00 micrograms/mg protein). Preoperative serum prolactin levels varied inversely with the prolactinoma calmodulin content (125, 257 and 3526 ng/ml, respectively). This study reveals that prolactinoma calmodulin content differs from normal, although it is not uniformly elevated as in other transformed tissues and that elevation of prolactinoma calmodulin content does not positively correlate with serum prolactin levels.

Syntheses are described for a range of N-(omega-aminoalkyl)-5-iodo- and -5-cyanonaphthalene-1-sulphonamides. The selective activity of these compounds as inhibitors for calmodulin-dependent phosphodiesterase (EC 3.1.4.17) is compared with their activity for the calmodulin-independent but calcium-dependent enzymes protein kinase C and transglutaminase (EC 2.3.2.13). The results show a drastic improvement in the selectivity of effect for the 5-iodo-compounds compared with the widely-used drug, W7, N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide.

The malaria parasite has an obligate calcium requirement for normal intracellular growth and invasion of host erythrocytes. Calmodulin (CaM) is a vital calcium-dependent protein present in eukaryotes. We found by radioimmunoassay that free parasites contain CaM. Schizont-infected erythrocytes had CaM levels of 23.3 +/- 2.7 ng per 10(6) cells compared to normals (11.2 +/- 1.5 ng per 10(6) cells). CaM levels were proportional to parasite maturity. Immunoelectron microscopy identified CaM diffusely within the cytoplasm of mature parasites and at the apical end of merozoites within the ductule of rhoptries, which may explain the calcium requirement for invasion. Cyclosporin A (CsA) was also found by electron microscopic autoradiography to concentrate in the food vacuole, as do chloroquine and mefloquine, and to distribute within the cytoplasm of mature parasites. The binding of dansylated CsA to schizont-infected erythrocytes was higher than to normal erythrocytes as analyzed by flow cytometry. Kinetic analysis revealed that binding was saturable for normal and infected erythrocytes and possibly free parasites. Competition for binding existed between dansylated CsA and native CsA as well as the CaM inhibitor W-7 and the classic antimalarial chloroquine. The in vitro growth of Plasmodium falciparum was sensitive to CaM antagonists, and in large part inhibition of the parasite was proportional to known anti-CaM potency. Antagonism existed between combinations of these drugs in multi-drug-resistant strains of P. falciparum, suggesting possible competition for the same binding site. In addition, the malaria parasite was also susceptible to calcium antagonists.

The effect of Ca2+ ion concentration on the 25 hydroxylation of tritiated cholecalciferol (3HD3) was investigated using homogenates of ovine liver from vitamin D replete sheep. A significant decrease in the production of 25 hydroxycholecalciferol (25OHD3) was observed when the concentration of Ca2+ in the homogenate was raised above 0.68 mmol/l by the addition of calcium gluconate. Similarly, a final concentration of 37 mumol EGTA/1 (equivalent to a Ca2+ concentration of 26.5 nmol/l) was associated with a 50% reduction of 25OHD3 production. That is, a broad bell-shaped relationship was observed between the production of 25OHD3 and the Ca2+ concentration in the homogenate. These changes in the rate of production of 25OHD3 were reproduced with hepatocytes from vitamin D replete rats, prepared by collagenase perfusion, using the drugs dantrolene sodium (DaNa) to reduce (ED50 = 57 mmol/l) and veratridine to increase (ED50 = 550 mmol/l) the intracellular Ca2+ concentration. Hepatocytes from vitamin D replete rats also showed a reduction in 25 hydroxylation of D3 (ED50 = 6 ng/ml) in response to the addition of 1-25 dihydroxycholecalciferol (1-25 (OH)2D3). The calmodulin antagonists; W7, compound 48/80, trifluoperazine (TFP) and calmidazolium (R24571) were all found to effect a dose response inhibition of the 25 hydroxylation of cholecalciferol by homogenates of ovine liver. R24571 had a similar inhibitory effect (ED50 = 70 mumol/l) upon the 25 hydroxylase enzyme of rat hepatocytes. It is concluded that the 25 hydroxylation of cholecalciferol in liver of vitamin D replete rats and sheep is calcium sensitive and is reduced in the presence of increased concentrations of 1,25(OH)2D3. Calmodulin may also be involved in the regulation of hepatocyte 25-hydroxylase activity by Ca2+.

Chromium at very low concentrations is an essential trace element--at higher concentrations it is associated with contact dermatitis and other toxicity problems. Its ionic radius is just outside that of other metal cations which have been found to activate calmodulin in vitro. We found that chromium was able to activate calmodulin at two different concentration ranges--over the micromolar range (which would probably never be achieved in man) a small degree of activation was found--but a much greater activation (76% of the maximum possible) was also found at nanomolar concentrations of chromium. In welders, who work with stainless steel and who were not reporting any physical symptoms of chromium toxicity, red cell chromium levels were 28.2 +/- 3.3 nM (n = 22) compared to 7.5 +/- 0.7 nM (n = 11) for normal controls. Thus, the concentration of chromium experienced within the cell can be of the order which will activate calmodulin in vitro. The possibility exists, therefore, that inappropriate activation of calmodulin could be relevant to chromium biology possibly contributing to the symptoms of chromium toxicity.

An assay for cyclic AMP phosphodiesterase in biological samples is described. The method is a continuous, spectrophotometric assay adapted for use on a centrifugal analyser. Using the calmodulin-activated cyclic AMP phosphodiesterase prepared from pig brain by DEAE-cellulose chromatography, and a standard calmodulin preparation from the same source, a rapid assay for calmodulin is described. The calmodulin assay has a working range of 2 to 6 mg/l calmodulin in the sample. The within-batch CV is less than 5% and the between-batch CV is 7.0% or less.

We have investigated the effects of the calmodulin antagonist trifluoperazine (TFP) on amylase secretion and adenosine 3':5'-monophosphate (cyclic AMP) metabolism using incubated parotid glands of young rats. Exposing unstimulated glands to 100 microM TFP doubled the basal rate of amylase and lactate dehydrogenase (LDH) release, but had no effect on either the parotid cyclic AMP or ATP contents. Isoprenaline (1 microM) stimulated amylase secretion and increased the tissue cyclic AMP content. 100 microM TFP inhibited these responses by 46% and 33%, respectively. N6,O2-dibutyryl adenosine 3',5'-monophosphate (dibutyryl cyclic AMP) mimicked the effect of isoprenaline on amylase release but 100 microM TFP had no effect on this response. 10 microM TFP inhibited F- -stimulated adenylate cyclase activity in a subcellular fraction isolated from the parotid by 32%. We conclude that TFP may inhibit isoprenaline-evoked amylase secretion from the rat parotid by an effect on either the catalytic or regulatory subunits of adenylate cyclase.

Bovine thyroid 100,000 X g supernatant contained calcium-activated, calmodulin-dependent protein kinase (PK-CaM) activity. The PK-CaM was partially purified using ion-exchange chromatography and characterized. PK-CaM, using casein as exogenous substrate, was not stimulated by Ca2+(0-500 microM) or calmodulin (1-10 micrograms) by themselves, but was stimulated by the combination of the two by 100%. The activation of the enzyme by Ca2+ and calmodulin was dose-dependent with maximal stimulation evident at 1 microM free-Ca2+ and 3 micrograms calmodulin. Both chlorpromazine and trifluoperazine inhibited the thyroid enzyme in a dose-related manner. The molecular weight (MW) of the PK-CaM, based on gel filtration, was approximately 500,000. PK-CaM could also be demonstrated using endogenous thyroid cytosol proteins as substrate. Separation of these 32P-labelled proteins by SDS-PAGE and subsequent autoradiography revealed that one major protein of approximately 56,000 MW was phosphorylated by PK-CaM. In some experiments, a second, less-intense protein band of approximately 64,000 MW was also phosphorylated. Evidence is presented, suggesting that these two protein bands may result from the autophosphorylation of the PK-CaM holoenzyme. These results offer a molecular mechanism, in addition to protein kinase C, by which Ca2+ effects may be mediated in thyroid.

A radioimmunoassay has been established and validated for the estimation of calmodulin levels in human peroral jejunal biopsy samples. Total levels were similar in biopsies from control subjects and patients with coeliac disease, both in relapse and remission. The subcellular distribution of calmodulin was determined in biopsy samples from control subjects and a patient with coeliac disease in relapse by rapid single-step analytical subcellular fractionation and immunoassay. In both situations most (greater than 70%) of the calmodulin was recovered in the cytosolic fractions with no selective enrichment in any single organelle.

Cell proliferation is often associated with an increase in calmodulin, the ubiquitous intracellular calcium receptor of non-muscle cells. A long lasting increase in the proliferative activity of brown adipose tissue is induced by cold exposure in the rat. The present work showed that this phenomenon is also associated with a rapid and long lasting increase in the calmodulin content of this tissue. It was equally shown that this increase can be reproduced by noradrenaline administration.

Both Ca2+ transport and binding properties of heart sarcolemmal membranes are altered in spontaneously hypertensive rats (SHR) when compared to their normotensive controls (WKY). The effects of calmodulin on these two processes were studied at free calcium concentrations presumed to be the physiological levels in the cytosol. At a calcium concentration of 2.10(-8)M, calmodulin did not significantly modify either binding or ATP-dependent accumulation of calcium by membranes of both origins. In contrast, at a free calcium concentration of 4.10(-7)M, calmodulin enhanced the calcium binding to SHR membranes and the ATP-dependent calcium transport by SHR and WKY membranes. Differences in calcium binding and ATP-dependent accumulation between the two substrains were suppressed in presence of calmodulin. These data demonstrate that modifications in calcium handling by SHR cardiac plasma membranes might be due to altered intracellular content or function of calmodulin in SHR.

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.

TSH (thyrotropin)-stimulated human thyroid adenylate cyclase has a biphasic response to Ca2+, being activated by submicromolar Ca2+ (optimum 22nM), with inhibition at higher concentrations. Calmodulin antagonists caused an inhibition of TSH-stimulated adenylate cyclase in a dose-dependent manner. Inhibition of TSH-and TSIg-(thyroid-stimulating immunoglobulins)-stimulated activity was more marked than that of basal, NaF- or forskolin-stimulated activity. This inhibition was not due to a decreased binding of TSH to its receptor. Addition of pure calmodulin to particulate preparations of human non-toxic goitre which had not been calmodulin-depleted had no effect on adenylate cyclase activity. EGTA was ineffective in removing calmodulin from particulate preparations, but treatment with the tervalent metal ion La3+ resulted in a loss of up to 98% of calmodulin activity from these preparations. Addition of La3+ directly to the adenylate cyclase assay resulted in a partial inhibition of TSH- and NaF-stimulated activity, with 50% inhibition produced by 5.1 microM and 4.0 microM-La3+ respectively. Particulate preparations with La3+ showed a decrease of TSH- and NaF-stimulated adenylate cyclase activity (approx. 40-60%). In La3+-treated preparations there was a decrease in sensitivity of TSH-stimulated adenylate cyclase to Ca2+ over a wide range of Ca2+ concentrations, but most markedly in the region of the optimal stimulatory Ca2+ concentration. In particulate preparations from which endogenous calmodulin had been removed by La3+ treatment, the addition of pure calmodulin caused an increase (73 +/- 22%; mean +/- S.E.M., n = 8) in TSH-stimulated thyroid adenylate cyclase activity. This was seen in 8 out of 13 experiments.

Calmodulin antagonists inhibited hormone-stimulated cyclic AMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare-earth-metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations was La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of calcium (approx. 10 nM) now failing to stimulate enzyme activity, high concentrations of calcium (greater than or equal to 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM-calmodulin in the presence of 10 microM free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 +/- 5% (mean +/- S.E.M., n = 7) of the activity in untreated, calmodulin-intact, preparations.

Human thyroid cells in monolayer responded to acute stimulation by TSH with an increase in the secretion of T3. This process appeared to be dependent on a rise in the cytosolic calcium concentration since the antagonist of intracellular calcium mobilization, TMB-8, was found to inhibit the release of T3 in response to TSH. The importance of intracellular calcium was further shown using the agent veratridine which increases the free calcium level within cells; veratridine potentiated the stimulation of T3 secretion by TSH and itself stimulated the release of T3 to a level higher than that seen in the presence of TSH alone. The calcium ionophore A23187 produced a biphasic effect on T3 secretion from human thyroid monolayers; at low concentrations, A23187 caused a decrease in both unstimulated and TSH-stimulated T3 secretion but above a concentration of 1 microM, T3 secretion was increased. The calmodulin antagonist W7 was found to inhibit T3 release in response to TSH, indicating a role for calmodulin in mediating the effects of intracellular calcium on T3 secretion.