Reduction Of Extracellular Calcium Research Articles

The sensitivity of mechanoelectrical transduction response phase to acoustic overstimulation is calcium-dependent

The Mechanoelectrical transduction (MET) channels of the mammalian hair cells are essential for converting sound stimuli into electrical signals that enable hearing. However, the impact of acoustic overstimulation, a leading cause of hearing loss, on the MET channel function remains poorly understood. In this study, I investigated the effect of loud sound-induced temporary threshold shift (TTS) on the transduction response phase across a wide range of sound frequencies and amplitudes. The results demonstrated an increase in the transduction response phase following TTS, indicating altered transduction apparatus function. Further investigations involving the reduction of extracellular calcium, a known consequence of TTS, replicated the observed phase changes. Additionally, reduction of potassium entry confirmed the specific role of calcium in regulating the transduction response phase. These findings provide novel insights into the impact of loud sound exposure on hearing impairment at the transduction apparatus level and highlight the critical role of calcium in modulating sound transduction. Considering that over 1 billion teenagers and young adults globally are at risk of hearing loss due to unsafe music listening habits, these results could significantly enhance awareness about the damaging effects of loud sound exposure.

Hypocalcemia-Induced Slowing of Human Sinus Node Pacemaking

Each heartbeat is initiated by cyclic spontaneous depolarization of cardiomyocytes in the sinus node forming the primary natural pacemaker. In patients with end-stage renal disease undergoing hemodialysis, it was recently shown that the heart rate drops to very low values before they suffer from sudden cardiac death with an unexplained high incidence. We hypothesize that the electrolyte changes commonly occurring in these patients affect sinus node beating rate and could be responsible for severe bradycardia. To test this hypothesis, we extended the Fabbri et al. computational model of human sinus node cells to account for the dynamic intracellular balance of ion concentrations. Using this model, we systematically tested the effect of altered extracellular potassium, calcium, and sodium concentrations. Although sodium changes had negligible (0.15 bpm/mM) and potassium changes mild effects (8 bpm/mM), calcium changes markedly affected the beating rate (46 bpm/mM ionized calcium without autonomic control). This pronounced bradycardic effect of hypocalcemia was mediated primarily by ICaL attenuation due to reduced driving force, particularly during late depolarization. This, in turn, caused secondary reduction of calcium concentration in the intracellular compartments and subsequent attenuation of inward INaCa and reduction of intracellular sodium. Our in silico findings are complemented and substantiated by an empirical database study comprising 22,501 pairs of blood samples and in vivo heart rate measurements in hemodialysis patients and healthy individuals. A reduction of extracellular calcium was correlated with a decrease of heartrate by 9.9 bpm/mM total serum calcium (p < 0.001) with intact autonomic control in the cross-sectional population. In conclusion, we present mechanistic in silico and empirical in vivo data supporting the so far neglected but experimentally testable and potentially important mechanism of hypocalcemia-induced bradycardia and asystole, potentially responsible for the highly increased and so far unexplained risk of sudden cardiac death in the hemodialysis patient population.

Cadherin Order and Dynamics in Calcium-Dependent and Independent Desmosomes

Desmosomes are complex cell-cell junctions composed of adhesive transmembrane cadherin proteins and intracellular plaque proteins. Integration of the desmosomal complex with the keratin intermediate filament cytoskeleton confers strength and mechanical resistance to epithelial tissue. Desmosomes can adopt different functional states in vivo that are critical for regulating adhesion during processes like wound healing and development. These states are characterized by desmosome sensitivity to calcium; upon reduction of extracellular calcium, cells with calcium-dependent desmosomes lose adhesion while cells with hyper-adhesive (calcium-independent) desmosomes maintain strong adhesion. Though defined by resistance to reduced calcium, the mechanism that confers hyper-adhesion is not known. Interestingly, the cadherin tertiary structure is dependent upon calcium binding for its rigidity. Furthermore, electron tomography showed desmosomal cadherins form an ordered array at the adhesive interface, and this order is hypothesized to be critical for adhesion. To address the mechanism driving hyper-adhesion, we investigated the order and dynamics of the desmosomal cadherin desmoglein 3 (Dsg3) in calcium dependent and hyper-adhesive states. Using fluorescence polarization microscopy, we found that Dsg3 was ordered in calcium-dependent desmosomes. When calcium was reduced, order was lost (τ= 5 min) faster than adhesive function was compromised (τ= 15 min). When we induced hyper-adhesion by inhibiting PKCα we similarly found Dsg3 was ordered. Surprisingly, when calcium was reduced in hyper-adhesive cells, order was lost with similar kinetics to the calcium-dependent cells, despite maintained adhesion. Measuring the order and dynamics of Dsg3 has revealed novel differences in ultrastructure between functional states in living cells. Our results demonstrate that desmosome adhesion is not directly dependent on cadherin order and cadherin trans-binding can persist after loss of tertiary structure. Because cadherins lose order regardless of adhesive state, these results suggest a central role for the desmosomal plaque in conferring hyper-adhesion.

Effects of Extracellular Calcium and 1,25 dihydroxyvitamin D3 on Sebaceous Gland Cells In vitro and In vivo

Calcium and 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) are promoters of epithelial cell functions; however their effects on sebaceous glands are unknown. In this study, morphology, ultrastructure, cell numbers, lipid synthesis and apoptosis of SZ95 sebocytes were assessed in vitro under different concentrations of extracellular calcium with or without 1,25(OH)2D3. Moreover, serum calcium and 1,25(OH)2D3 levels were assessed in acne and non-acne patients (controls). Under conditions of low extracellular calcium, lipogenesis and cell detachment were observed. Increasing extracellular calcium enhanced sebocyte numbers, induced epithelial morphology and reduced lipogenesis. Moreover, a reduction in extracellular calcium reduced E-cadherin and enhanced caspase 3/7 activity (apoptosis), whereas calcium chelation by EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) resulted in enhanced lipogenesis. 1,25(OH)2D3 decreased sebaceous lipogenesis, but also induced signs of autophagy. In the clinical study, patients and controls exhibited normal serum calcium levels. Younger acne patients presented lower 1,25(OH)2D3 levels than did older ones. In conclusion, extracellular calcium and 1,25(OH)2D3 regulate sebocyte morphology, increase cell numbers, decrease sebaceous lipogenesis and induce cell autophagy in vitro. The increased ionized calcium and the reduced 1,25(OH)2D3 levels detected in the serum of younger patients with acne may contribute respectively to increased sebaceous gland volume and enhanced lipogenesis.

Neocortical GABA release at high intracellular sodium and low extracellular calcium: an anti-seizure mechanism.

In epilepsy, the GABA and glutamate balance may be disrupted and a transient decrease in extracellular calcium occurs before and during a seizure. Flow Cytometry based fluorescence activated particle sorting experiments quantified synaptosomes from human neocortical tissue, from both epileptic and non-epileptic patients (27.7% vs. 36.9% GABAergic synaptosomes, respectively). Transporter-mediated release of GABA in human and rat neocortical synaptosomes was measured using the superfusion technique for the measurement of endogenous GABA. GABA release was evoked by either a sodium channel activator or a sodium/potassium-ATPase inhibitor when exocytosis was possible or prevented, and when the sodium/calcium exchanger was active or inhibited. The transporter-mediated release of GABA is because of elevated intracellular sodium. A reduction in the extracellular calcium increased this release (in both non-epileptic and epileptic, except Rasmussen encephalitis, synaptosomes). The inverse was seen during calcium doubling. In humans, GABA release was not affected by exocytosis inhibition, that is, it was solely transporter-mediated. However, in rat synaptosomes, an increase in GABA release at zero calcium was only exhibited when the exocytosis was prevented. The absence of calcium amplified the sodium/calcium exchanger activity, leading to elevated intracellular sodium, which, together with the stimulation-evoked intracellular sodium increment, enhanced GABA transporter reversal. Sodium/calcium exchange inhibitors diminished GABA release. Thus, an important seizure-induced extracellular calcium reduction might trigger a transporter- and sodium/calcium exchanger-related anti-seizure mechanism by augmenting transporter-mediated GABA release, a mechanism absent in rats. Uniquely, the additional increase in GABA release because of calcium-withdrawal dwindled during the course of illness in Rasmussen encephalitis. Seizures cause high Na(+) influx through action potentials. A transient decrease in [Ca(2+)]e (seizure condition) increases GABA transporter (GAT)-mediated GABA release because of elevated [Na(+)]i. This amplifies the Sodium-Calcium-Exchanger (NCX) activity, further increasing [Na(+)]i and GABA release. The reduction in [Ca(2+)]e triggers a GAT-NCX related anti-seizure mechanism by augmenting GAT-mediated GABA release. This mechanism, obvious in humans, is absent in rats.

Modulation of microglial process convergence toward neuronal dendrites by extracellular calcium.

Extracellular calcium concentrations in the brain fluctuate during neuronal activities and may affect the behavior of brain cells. Microglia are highly dynamic immune cells of the brain. However, the effects of extracellular calcium concentrations on microglial dynamics have not been investigated. Here, we addressed this question in mouse brain slices and in vivo using two-photon microscopy. We serendipitously found that extracellular calcium reduction induced microglial processes to converge at distinct sites, a phenomenon we termed microglial process convergence (MPCs). Our studies revealed that MPCs target neuronal dendrites independent of neuronal action potential firing and is mediated by ATP release and microglial P2Y12 receptors. These results indicate that microglia monitor and interact with neurons during conditions of cerebral calcium reduction in the normal and diseased brain.

Bupivacaine Induces Short-Term Alterations and Impairment in Rat Tendons

Background: Toxicity of the local anesthetic bupivacaine (BV) has been a matter of debate across medical fields. Numerous in vitro studies demonstrate considerable toxicity of BV on various cell types. Purpose: This study addresses the question of how tendon tissue responds to BV in vivo and in vitro. Study Design: Controlled laboratory study. Methods: In vitro studies on cultured rat Achilles tendon–derived cells were performed with cell viability assays and cleaved caspase 3 immunocytochemistry. Quantitative reverse transcription–polymerase chain reaction, Western blotting, gelatin zymography, and a biomechanical testing routine were applied on rat Achilles tendons at 1 and 4 weeks after a single unilateral peritendinous injection of 0.5% BV. The BV-mediated cell death in tendons was estimated with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and immunohistochemical detection of cleaved caspase 3. Results: Treatment of rat tendon–derived cells with 0.5% bupivacaine for 10 minutes had detrimental effects on cell viability, which can be reduced by N-acetyl-L-cysteine or reduction of extracellular calcium. In vivo, single peritendinous injections of BV caused apoptosis in endotenon cells and an increase of pro–matrix metalloproteinase-9 after 6 hours. The collagen ratio shifted toward collagen type III after 6 hours and 2 days; scleraxis messenger RNA (mRNA) expression was reduced by 87%. Maximum tensile load was reduced by 17.6% after 1 week. Conclusion: Bupivacaine exerts a severe, reactive oxygen species–mediated effect on tendon cell viability in vitro in a time- and dose-dependent manner, depending on extracellular calcium concentration. Culture conditions need to be taken into account when in vitro data are translated into the in vivo situation. In vivo, administration of BV elicits a marked but temporary functional damage. Clinical Relevance: Local anesthetics cause short-term alterations in rat tendons, which, if occurring in humans to a similar extent, may be relevant regarding decreased biomechanical properties and increased vulnerability to tendon overload or injury.

Reversal of Dopamine Inhibition of Dopaminergic Neurons of the Ventral Tegmental Area is Mediated by Protein Kinase C

Adaptation of putative dopaminergic (pDA) neurons in the ventral tegmental area (VTA) to drugs of abuse may alter information processing related to reward and reinforcement and is an important factor in the development of addiction. We have demonstrated that prolonged increases in the concentration of dopamine (DA) result in a time-dependent decrease in sensitivity of pDA neurons to DA, which we termed DA inhibition reversal (DIR). In this study, we used extracellular recordings to examine factors mediating DIR. A 40 min administration of DA (2.5-10 μM), but not the DA D2 receptor agonist quinpirole (50-200 nM), resulted in inhibition of neuronal firing followed by DIR. In the presence of 100 nM cocaine, inhibition followed by DIR was seen with much lower DA concentrations. Reversal of quinpirole inhibition could be induced by an activator of protein kinase C, but not of protein kinase A. Inhibitors of protein kinase C or phospholipase C blocked the development of DIR. Disruption of intracellular calcium release also prevented DIR. Reduction of extracellular calcium or inhibition of store-operated calcium entry blocked DIR, but the L-type calcium channel blocker nifedipine did not. DIR was age-dependent and not seen in pDA VTA neurons from rat pups younger than 15 days postnatally. Our data indicate that DIR is mediated by protein kinase C, and implicate a conventional protein kinase C. This characterization of DIR gives insight into the regulation of autoinhibition of pDA VTA neurons, and the resulting long-term alteration in information processing related to reward and reinforcement.

Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue

Background. Streptococcus pneumoniae causes serious diseases such as pneumonia and meningitis. Its major pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, which produces lytic pores at high concentrations. At low concentrations, it has other effects, including induction of apoptosis. Many cellular effects of pneumolysin appear to be calcium dependent.Methods. Live imaging of primary mouse astroglia exposed to sublytic amounts of pneumolysin at various concentrations of extracellular calcium was used to measure changes in cellular permeability (as judged by lactate dehydrogenase release and propidium iodide chromatin staining). Individual pore properties were analyzed by conductance across artificial lipid bilayer. Tissue toxicity was studied in continuously oxygenated acute brain slices.Results. The reduction of extracellular calcium increased the lytic capacity of the toxin due to increased membrane binding. Reduction of calcium did not influence the conductance properties of individual toxin pores. In acute cortical brain slices, the reduction of extracellular calcium from 2 to 1 mM conferred lytic activity to pathophysiologically relevant nonlytic concentrations of pneumolysin.Conclusions. Reduction of extracellular calcium strongly enhanced the lytic capacity of pneumolysin due to increased membrane binding. Thus, extracellular calcium concentration should be considered as a factor of primary importance for the course of pneumococcal meningitis.

Proteinase-Activated Receptors 1 and 2 Exert Opposite Effects on Renal Renin Release

Proteinase-activated receptors (PARs) 1 to 4 are highly expressed in the kidney and are involved in the regulation of renal hemodynamics and tubular function. Since intravascular infusion of the proteinase thrombin, which activates PARs, has been shown to decrease plasma renin activity in rats, we investigated the effects of the respective PAR subtypes on renin release using the isolated perfused mouse kidney model. Thrombin dose-dependently reduced perfusate flow and inhibited renin secretion rates (RSRs) that had been prestimulated by the β-adrenoreceptor agonist isoproterenol. The suppression of RSRs was prevented by the selective PAR1 inhibitor SCH79797, and direct activation of PAR1 by TFLLR mimicked the effects of thrombin on RSRs and vascular tone. Moreover, TFLLR suppressed the stimulations of RSRs in response to the loop diuretic bumetanide, to prostaglandin E(2), or to a decrease in renal perfusion pressure but not in response to a reduction in extracellular calcium. The PAR2-activating peptide SLIGRL concentration dependently increased RSR and perfusate flow. The stimulation of RSRs by SLIGRL was markedly attenuated by N(G)-nitro-L-arginine methyl ester, suggesting an NO-dependent mechanism. Activation of PAR4 by AYPGKF did not modulate RSRs or perfusate flow. PAR1 and PAR2 immunoreactivity were detected in the juxtaglomerular region and were colocalized with renin immunoreactivity. Our data provide evidence that PAR1 activation inhibits renal renin secretion and induces renal vasoconstriction, whereas PAR2 activation stimulates renin release and induces vasodilation mainly via the release of NO.

Shear stress-induced volume decrease in C11-MDCK cells by BK-α/β4

Large-conductance, calcium-activated potassium channels (BK) are expressed in principal cells (PC) and intercalated cells (IC) in mammalian nephrons as BK-alpha/beta1 and BK-alpha/beta4, respectively. IC, which protrude into the lumens of tubules, express substantially more BK than PC despite lacking sufficient Na-K-ATPase to support K secretion. We previously showed in mice that IC exhibit size reduction when experiencing high distal flows induced by a high-K diet. We therefore tested the hypothesis that BK-alpha/beta4 are regulators of IC volume via a shear stress (tau)-induced, calcium-dependent mechanism, resulting in a reduction in intracellular K content. We determined by Western blot and immunocytochemical analysis that C11-Madin-Darby canine kidney cells contained a predominance of BK-alpha/beta4. To determine the role of BK-alpha/beta4 in tau-induced volume reduction, we exposed C11 cells to tau and measured K efflux by flame photometry and cell volume by calcein staining, which changes inversely to cell volume. With 10 dynes/cm(2), calcein intensity significantly increased 39% and monovalent cationic content decreased significantly by 37% compared with static conditions. Furthermore, the shear-induced K loss from C11 was abolished by the reduction of extracellular calcium, addition of 5 mM TEA, or BK-beta4 small interfering (si) RNA, but not by addition of nontarget siRNA. These results show that BK-alpha/beta4 plays a role in shear-induced K loss from IC, suggesting that BK-alpha/beta4 regulate IC volume during high-flow conditions. Furthermore, these results support the use of C11 cells as in vitro models for studying BK-related functions in IC of the kidney.

Effects of the NO donor sodium nitroprusside on oxygen consumption and energetics in rabbit myocardium

Nitric oxide (NO) has influence on various cellular functions. Little is known of the influence of NO on myocardial energetics. In the present study oxygen consumption and mechanical parameters of isometrically contracting rabbit papillary muscles (1 Hz stimulation frequency) were investigated at varying interventions while maintaining physiological conditions (37°C; 2.5 mM Ca2+) to study the effects of NO on energetics. The NO donor sodium nitroprusside (SNP) showed a negative inotropic effect. SNP decreased the maximal force in normal rabbit muscle strips by 30%, the force time integral (FTI) by 40% and the relaxation time by 20%. In addition the oxygen consumption decreased by 60%, a notably disproportional decrease compared to the mechanical parameters. Consequently, the economy as a ratio of FTI and oxygen consumption is significantly increased by SNP. In contrast the negative inotropic effect due to a reduction in extracellular Calcium (Ca2+) from 2.5 to 1.25 mM reduced FTI and oxygen consumption proportionally by 40% and did not change economy. The effect of NO on force and oxygen consumption could be reproduced by the application of the cyclic guanosine monophosphate (cGMP) analogue 8-bromo-cGMP. In summary, NO increased the economy of isometrically contracting papillary muscles. The improvement in contraction economy under NO seems to be mediated by cGMP as the secondary messenger and maybe due to alterations of the crossbridge cycle.

Effect of calcium-sensing receptor activation in models of autosomal recessive or dominant polycystic kidney disease

Background. Antagonists of relevant Gs protein-coupled and agonists of relevant Gi protein-coupled receptors lower renal cAMP and inhibit growth of renal cysts in animal models of human ARPKD (PCK rat) and/or ADPKD (Pkd2−/WS25 mouse). A calcium-sensing receptor (CaR) is expressed in various tubular segments and couples to Gq, thereby activating phospholipase Cγ, InsP3 generation and calcium mobilization from intracellular stores, and Gi proteins. By both mechanisms, CaR activation could lower intracellular cAMP and inhibit renal cyst growth.Methods. PCK rat and Pkd2−/WS25 mouse littermates were fed rodent chow without or with R-568, a type 2 calcimimetic, at a concentration of 0.05% or 0.1% between 3 and 10 or 16 weeks of age. Histomorphometric analysis was performed with Meta-Morph software. Western analysis and immunohistochemical staining were performed using antibodies for aquaporin-2, urea transporter UT-A1 and CaR. Northern blot hybridization was used to quantify the expression of vasopressin V2 receptor and aquaporin 2 mRNAs. Cyclic AMP was measured using an enzyme immunoassay kit.Results. R-568 had no effect on kidney weight, cyst volume, plasma BUN concentration or severity of the polycystic liver disease. A significant reduction in renal interstitial fibrosis was detected in PCK rats, but not in Pkd2−/WS25 mice. R-568 administration, as anticipated, resulted in hypocalcemia and hyperphosphatemia, and significant increases in urine output, osmolar clearance, and urinary excretions of sodium, potassium and calcium.Conclusions. CaR activation had no detectable effect on cystogenesis in models of autosomal recessive or dominant polycystic kidney disease. The lack of protective effect could be due to the absence of CaR in the outer medullary and cortical collecting ducts, the reduction in extracellular calcium and the unaffected levels of renal cAMP and renal expression of cAMP-dependent genes. A possible beneficial effect on interstitial fibrosis deserves further study at more advanced stages of the disease.

An Innexin-Dependent Cell Network Establishes Left-Right Neuronal Asymmetry in C. elegans

Gap junctions are widespread in immature neuronal circuits, but their functional significance is poorly understood. We show here that a transient network formed by the innexin gap-junction protein NSY-5 coordinates left-right asymmetry in the developing nervous system of Caenorhabditis elegans. nsy-5 is required for the left and right AWC olfactory neurons to establish stochastic, asymmetric patterns of gene expression during embryogenesis. nsy-5-dependent gap junctions in the embryo transiently connect the AWC cell bodies with those of numerous other neurons. Both AWCs and several other classes of nsy-5-expressing neurons participate in signaling that coordinates left-right AWC asymmetry. The right AWC can respond to nsy-5 directly, but the left AWC requires nsy-5 function in multiple cells of the network. NSY-5 forms hemichannels and intercellular gap-junction channels in Xenopus oocytes, consistent with a combination of cell-intrinsic and network functions. These results provide insight into gap-junction activity in developing circuits.

Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells

Cardiotonic glycosides, like ouabain, inhibit Na(+)-K(+)-ATPase. Recent evidence suggests that low molar concentrations of ouabain alter cell growth. Studies were conducted to examine the effect of ouabain on Akt phosphorylation and rate of cell proliferation in opossum kidney (OK) proximal tubule cells. Cells exposed to 10 nM ouabain displayed increased Akt Ser(473) phosphorylation, as evidenced by an increase in phospho-Akt Ser(473) band density. Ouabain-stimulated Akt Ser(473) phosphorylation was inhibited by pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and wortmannin), a PLC inhibitor (edelfosine), and an Akt inhibitor. Moreover, ouabain-mediated Akt Ser(473) phosphorylation was suppressed by reduction of extracellular calcium (EGTA) or when intracellular calcium was buffered by BAPTA-AM. An inhibitor of calcium store release (TMB-8) and an inhibitor of calcium entry via store-operated calcium channels (SKF96365) also suppressed ouabain-mediated Akt Ser(473) phosphorylation. In fura-2 AM-loaded cells, 10 nM ouabain increased capacitative calcium entry (CCE). Ouabain at 10 nM did not significantly alter baseline cytoplasmic calcium concentration in control cells. However, treatment with 10 nM ouabain caused a significantly higher ATP-mediated calcium store release. After 24 h, 10 nM ouabain increased the rate of cell proliferation. The Akt inhibitor, BAPTA-AM, SKF96365, and cyclopiazonic acid suppressed the increase in the rate of cell proliferation caused by 10 nM ouabain. Ouabain at 10 nM caused a detectable increase in (86)Rb uptake but did not significantly alter Na(+)-K(+)-ATPase (ouabain-sensitive pNPPase) activity in crude membranes or cell sodium content. Taken together, the results point to a role for CCE and Akt phosphorylation, in response to low concentrations of ouabain, that increase the rate of cell proliferation without inhibiting Na(+)-K(+)-ATPase-mediated ion transport.

Flow-mediated dilatation in human circulation: diagnostic and therapeutic aspects

a disturbance of endothelial function is considered as a key event in the development of atherosclerosis ([63][1]). Thus reliable assessment of endothelial function in humans appears highly desirable. With respect to the major endothelial functions, this aim can be achieved by different approaches:

A mathematical/physiological model of parathyroid hormone secretion in response to blood-ionized calcium lowering in vivo

The aim of the present study was to test a mathematical model of the biochemical processes in the parathyroid glands responsible for the secretion of parathyroid hormone resulting from extracellular calcium reduction. A double exponential curve described the parathyroid hormone secretion induced by rapid lowering of blood-ionized calcium in humans with normal as well as abnormal parathyroid tissue. Our data show that it was possible to establish a simple mathematical model of the parathyroid hormone response to blood-ionized calcium lowering, sufficient to fit experimental data obtained from patients with abnormal and normal parathyroid tissue. The fitted parameters showed no significant differences between patients with insulin-dependent diabetes mellitus and controls. In primary hyperparathyroidism, the parathyroid hormone production and steady-state transport across the cell membrane were increased, probably due to the larger amount of parathyroid tissue in these patients. These observations reveal a striking functional similarity between abnormal and normal parathyroid tissue. Furthermore, an apparently linear relationship between the rate of parathyroid hormone elimination from the blood plasma and the rate of cellular production/secretion was observed. This could be interpreted as an adaptation of the parathyroid gland's ability to produce parathyroid hormone depending on the average demand from the body.

Gap Junction Formation between Cultured Embryonic Lens Cells Is Inhibited by Antibody to N-Cadherin

Intercellular communication mediated by gap junctions is important for tissue homeostasis in the avascular lens, and extensive areas of gap junctions form between fiber cells during fiber cell differentiation and lens development. We examined the role of the calcium-dependent cell adhesion molecule, N-cadherin, in the process of gap junction formation between fiber cells. Lentoids, multicellular structures with characteristics of differentiated fiber cells, were isolated from embryonic chick lens cultures and subsequently paired to provide anin vitromodel of fiber cell interactions. Gap junction formation between cells of paired lentoids was monitored by observing the lentoid-to-lentoid transfer of fluorescent dyes, either calcein or Lucifer yellow, over a time course of up to 48 hr. Dye transfer between lentoids was inhibited upon the addition to the medium of Fab fragments (100–622 μg/ml) of a monoclonal antibody specific for N-cadherin, and also by the reduction of extracellular calcium in the incubation medium. However, the addition of Fab fragments (100–1500 μg/ml) of an antibody to a fiber-cell-specific integral membrane protein, MIP, did not change the time course nor extent of dye transfer between lentoids. Our results, using cultured embryonic cells, extend those from previous studies with cell lines and transfected cells. We conclude that cadherin interactions facilitate the formation of gap junctions between embryonic lens fiber cells, by the stabilization of membrane appositions and/or by the generation of an intracellular signal(s).

Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells.

During the assembly of gap junctions, a hemichannel in the plasma membrane of one cell is thought to align and dock with another in an apposed membrane to form a cell-to-cell channel. We report here on the existence and properties of nonjunctional, plasma membrane connexin43 (Cx43) hemichannels. The opening of the hemichannels was demonstrated by the cellular uptake of 5(6)-carboxyfluorescein from the culture medium when extracellular calcium levels were reduced. Dye uptake exhibited properties similar to those of gap junction channels. For example, using different dyes, the levels of uptake were correlated with molecular size: 5(6)-carboxyfluorescein (approximately 32%), 7-hydroxycoumarin-3-carboxylic acid (approximately 24%), fura-2 (approximately 11%), and fluorescein-dextran (approximately 0.4%). Octanol and heptanol also reduced dye uptake by approximately 50%. Detailed analysis of one clone of Novikoff cells transfected with a Cx43 antisense expression vector revealed a reduction in dye uptake levels according to uptake assays and a corresponding decrease in intercellular dye transfer rates in microinjection experiments. In addition, a more limited decrease in membrane resistance upon reduction of extracellular calcium was detected in electrophysiological studies of antisense transfectants, in contrast to control cells. Studies of dye uptake in HeLa cells also demonstrated a large increase following transfection with Cx43. Together these observations indicate that Cx43 is responsible for the hemichannel function in these cultured cells. Similar dye uptake results were obtained with normal rat kidney (NRK) cells, which express Cx43. Dye uptake can be dramatically inhibited by 12-O-tetradeconylphorbol-13-acetate-activated protein kinase C in these cell systems and by a temperature-sensitive tyrosine protein kinase, pp60v-src in LA25-NRK cells. We conclude that Cx43 hemichannels are found in the plasma membrane, where they are regulated by multiple signaling pathways, and likely represent an important stage in gap junction assembly.

Inhibition of In Vitro Meningioma Proliferation after Growth Factor Stimulation by Calcium Channel Antagonists

We have previously reported that calcium channel antagonists can block both the growth of meningiomas in culture and the potent growth stimulation of meningioma cells by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). This study further defines the nature of this growth inhibition. Primary meningioma cultures were established, and cells were characterized. Fibroblast growth factor or insulin-like growth factor-I growth stimulation in the presence of calcium channel antagonists was examined. In addition, the effects of ethylene glycol-bis-(aminoethylether) N,N,N',N"-tetraacetic acid and Bay K 8644, a calcium channel agonist, on the growth factors were analyzed. Growth factor receptor immunohistochemistry was performed on the original tumors and the in vitro meningioma cells. Twelve of 17 (71%) meningiomas in this study were positive for the EGF receptor, and 14 of 17 (82%) were positive for the PDGF receptor. Five of six (83%) of the culture cells were positive for the EGF receptor, and four of five (80%) were positive for the PDGF receptor. Intracellular calcium changes were quantified using the intracellular calcium-chelating, fluorescent dye, Fura-2. The growth stimulation of fibroblast growth factor and insulin-like growth factor-I on meningioma cells in culture was decreased in a dose-dependent manner by calcium channel antagonists. The growth stimulation of fibroblast growth factor and insulin-like growth factor-I was not affected by a reduction of extracellular calcium, whereas the growth stimulation of EGF and PDGF was. Interestingly, intracellular calcium was not increased after exposure to growth factors but was increased after serum stimulation. This increase could be blocked by preincubation with verapamil. Calcium channel antagonists can inhibit proliferation of meningioma cells in culture after stimulation with a number of growth factors. These drugs might disrupt intracellular calcium homeostasis or interfere with key elements of the growth factor signal transduction pathways. These mechanisms as well as the potential clinical relevance of these findings are discussed.