Integrin mobilizes intracellular Ca(2+) in renal vascular smooth muscle cells

Peptides with the Arg-Gly-Asp (RGD) motif induce vasoconstriction in rat afferent arterioles by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)) in vascular smooth muscle cells (VSMC). This finding suggests that occupancy of integrins on the plasma membrane of VSMC might affect vascular tone. The purpose of this study was to determine whether occupancy of integrins by exogenous RGD peptides initiates intracellular Ca(2+) signaling in cultured renal VSMC. When smooth muscle cells were exposed to 0.1 mM hexapeptide GRGDSP, [Ca(2+)](i) rapidly increased from 91 +/- 4 to 287 +/- 37 nM and then returned to the baseline within 20 s (P < 0.05, 34 cells/5 coverslips). In controls, the hexapeptide GRGESP did not trigger Ca(2+) mobilization. Local application of the GRGDSP induced a regional increase of cytoplasmic [Ca(2+)](i), which propagated as Ca(2+) waves traveling across the cell and induced a rapid elevation of nuclear [Ca(2+)](i). Spontaneous recurrence of smaller-amplitude Ca(2+) waves were found in 20% of cells examined after the initial response to RGD-containing peptides. Blocking dihydropyridine-sensitive Ca(2+) channels with nifedipine or removal of extracellular Ca(2+) did not inhibit the RGD-induced Ca(2+) mobilization. However, pretreatment of 20 microM ryanodine completely eliminated the RGD-induced Ca(2+) mobilization. Anti-beta(1) and anti-beta(3)-integrin antibodies with functional blocking capability simulate the effects of GRGDSP in [Ca(2+)](i). Incubation with anti-beta(1)- or beta(3)-integrin antibodies inhibited the increase in [Ca(2+)](i) induced by GRGDSP. We conclude that exogenous RGD-containing peptides induce release of Ca(2+) from ryanodine-sensitive Ca(2+) stores in renal VSMC via integrins, which can trigger cytoplasmic Ca(2+) waves propagating throughout the cell.     

Platelet-activating factor-induced homologous and heterologous desensitization in cultured vascular smooth muscle cells

Intracellular free Ca2+ concentrations were monitored in vascular smooth muscle cells (VSMC) using the Ca2+-sensitive dye fura II. Superfusion of VSMC with platelet-activating factor (S-PAF; 1-100 nM) increased cytosolic Ca2+ in a dose-dependent manner. The response was transient and returned to base line even though the agonist was still present. A second, higher dose of PAF did not elicit a response. The inactive optical isomer, R-PAF, was ineffective suggesting that the S-PAF response is specific and receptor-mediated. Pretreatment of VSMC with PAF attenuated angiotensin II-stimulated Ca2+ mobilization but not vasopressin-stimulated Ca2+ mobilization. Treatment of VSMC with PAF (10 nM) stimulated inositol trisphosphate and inositol tetrakisphosphate formation above control by 260 +/- 15% and 195 +/- 11%, respectively. Diacylglycerol levels also rose during PAF stimulation and remained increased over 15 min. Pretreatment of VSMCs with phorbol-12,13-myristate acetate (10 nM) for 30 min abolished both the PAF- and angiotensin II-induced increases in cytosolic Ca2+, but not the vasopressin-induced increase. Pretreatment of VSMC with dioctanoylglycerol (10 microM) abolished the S-PAF-, angiotensin II-, and vasopressin-induced elevation in cytosolic Ca2+. We propose that this desensitization is possibly mediated by diacylglycerol formed in response to PAF.     

Effects of Ca2+ channel antagonists on nerve stimulation-induced and ischemia-induced myocardial interstitial acetylcholine release in cats

Although an axoplasmic Ca(2+) increase is associated with an exocytotic acetylcholine (ACh) release from the parasympathetic postganglionic nerve endings, the role of voltage-dependent Ca(2+) channels in ACh release in the mammalian cardiac parasympathetic nerve is not clearly understood. Using a cardiac microdialysis technique, we examined the effects of Ca(2+) channel antagonists on vagal nerve stimulation- and ischemia-induced myocardial interstitial ACh releases in anesthetized cats. The vagal stimulation-induced ACh release [22.4 nM (SD 10.6), n = 7] was significantly attenuated by local administration of an N-type Ca(2+) channel antagonist omega-conotoxin GVIA [11.7 nM (SD 5.8), n = 7, P = 0.0054], or a P/Q-type Ca(2+) channel antagonist omega-conotoxin MVIIC [3.8 nM (SD 2.3), n = 6, P = 0.0002] but not by local administration of an L-type Ca(2+) channel antagonist verapamil [23.5 nM (SD 6.0), n = 5, P = 0.758]. The ischemia-induced myocardial interstitial ACh release [15.0 nM (SD 8.3), n = 8] was not attenuated by local administration of the L-, N-, or P/Q-type Ca(2+) channel antagonists, by inhibition of Na(+)/Ca(2+) exchange, or by blockade of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptor but was significantly suppressed by local administration of gadolinium [2.8 nM (SD 2.6), n = 6, P = 0.0283]. In conclusion, stimulation-induced ACh release from the cardiac postganglionic nerves depends on the N- and P/Q-type Ca(2+) channels (with a dominance of P/Q-type) but probably not on the L-type Ca(2+) channels in cats. In contrast, ischemia-induced ACh release depends on nonselective cation channels or cation-selective stretch activated channels but not on L-, N-, or P/Q type Ca(2+) channels, Na(+)/Ca(2+) exchange, or Ins(1,4,5)P(3) receptor-mediated pathway.     

Nonsteroidal anti-inflammatory drugs inhibit vascular smooth muscle cell proliferation by enabling the Ca2+-dependent inactivation of calcium release-activated calcium/orai channels normally prevented by mitochondria

Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to occlusive and proliferative disorders of the vessel wall. Salicylate and other nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit VSMC proliferation by an unknown mechanism unrelated to anti-inflammatory activity. In search for this mechanism, we have studied the effects of salicylate and other NSAIDs on subcellular Ca(2+) homeostasis and Ca(2+)-dependent cell proliferation in rat aortic A10 cells, a model of neointimal VSMCs. We found that A10 cells displayed both store-operated Ca(2+) entry (SOCE) and voltage-operated Ca(2+) entry (VOCE), the former being more important quantitatively than the latter. Inhibition of SOCE by specific Ca(2+) released-activated Ca(2+) (CRAC/Orai) channels antagonists prevented A10 cell proliferation. Salicylate and other NSAIDs, including ibuprofen, indomethacin, and sulindac, inhibited SOCE and thereby Ca(2+)-dependent, A10 cell proliferation. SOCE, but not VOCE, induced mitochondrial Ca(2+) uptake in A10 cells, and mitochondrial depolarization prevented SOCE, thus suggesting that mitochondrial Ca(2+) uptake controls SOCE (but not VOCE) in A10 cells. NSAIDs depolarized mitochondria and prevented mitochondrial Ca(2+) uptake, suggesting that they favor the Ca(2+)-dependent inactivation of CRAC/Orai channels. NSAIDs also inhibited SOCE in rat basophilic leukemia cells where mitochondrial control of CRAC/Orai is well established. NSAIDs accelerate slow inactivation of CRAC currents in rat basophilic leukemia cells under weak Ca(2+) buffering conditions but not in strong Ca(2+) buffer, thus excluding that NSAIDs inhibit SOCE directly. Taken together, our results indicate that NSAIDs inhibit VSMC proliferation by facilitating the Ca(2+)-dependent inactivation of CRAC/Orai channels which normally is prevented by mitochondria clearing of entering Ca(2+).     

Metabotropic Ca2+ channel-induced calcium release in vascular smooth muscle

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca(2+)] owing to either Ca(2+) influx through voltage-gated Ca(2+) channels of the plasmalemma or to receptor-mediated Ca(2+) release from the sarcoplasmic reticulum (SR). Although the ionotropic role of L-type Ca(2+) channels is well known, we review here data suggesting a new role of these channels in arterial myocytes. After sensing membrane depolarization Ca(2+) channels activate G proteins and the phospholipase C/inositol 1,4,5-trisphosphate (InsP(3)) pathway. Ca(2+) released through InsP(3)-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca(2+) signal, thus triggering arterial cerebral vasoconstriction in the absence of extracellular calcium influx. This metabotropic action of L-type Ca(2+) channels, denoted as calcium channel-induced Ca(2+) release, could have implications in cerebral vascular pharmacology and pathophysiology, because it can be suppressed by Ca(2+) channel antagonists and potentiated with small concentrations of extracellular vasoactive agents as ATP.     

Integrin-mediated mechanotransduction in renal vascular smooth muscle cells: activation of calcium sparks

Integrins are transmembrane heterodimeric proteins that link extracellular matrix (ECM) to cytoskeleton and have been shown to function as mechanotransducers in nonmuscle cells. Synthetic integrin-binding peptide triggers Ca(2+) mobilization and contraction in vascular smooth muscle cells (VSMCs) of rat afferent arteriole, indicating that interactions between the ECM and integrins modulate vascular tone. To examine whether integrins transduce extracellular mechanical stress into intracellular Ca(2+) signaling events in VSMCs, unidirectional mechanical force was applied to freshly isolated renal VSMCs through paramagnetic beads coated with fibronectin (natural ligand of alpha(5)beta(1)-integrin in VSMCs). Pulling of fibronectin-coated beads with an electromagnet triggered Ca(2+) sparks, followed by global Ca(2+) mobilization. Paramagnetic beads coated with low-density lipoprotein, whose receptors are not linked to cytoskeleton, were minimally effective in triggering Ca(2+) sparks and global Ca(2+) mobilization. Preincubation with ryanodine, cytochalasin-D, or colchicine substantially reduced the occurrence of Ca(2+) sparks triggered by fibronectin-coated beads. Binding of VSMCs with antibodies specific to the extracellular domains of alpha(5-) and beta(1)-integrins triggered Ca(2+) sparks simulating the effects of fibronectin-coated beads. Preincubation of microperfused afferent arterioles with ryanodine or integrin-specific binding peptide inhibited pressure-induced myogenic constriction. In conclusion, integrins transduce mechanical force into intracellular Ca(2+) signaling events in renal VSMCs. Integrin-mediated mechanotransduction is probably involved in myogenic response of afferent arterioles.     

A functional study on L-type calcium channels in granulosa cells of small follicles in laying and forced molt hens

To investigate Ca(2+) dynamics in earlier phases of follicular development we compared the resting [Ca(2+)](i) and tested the functional responses to agonist/antagonist of L-type voltage-operated calcium channels (VOCCs) in small follicles GCs from hens during oviposition (O-GCs) and forced molt (M-GCs), using the microspectrofluorimetric [Ca(2+)](i) imaging. O-GCs were obtained from prehierarchical follicles (F(6)-F(5)-F(4)<8mm). In basal and agonist/antagonist stimulated M-GCs we did not observe a change in the [Ca(2+)](i) under any of condition in all cells analyzed. Based on basal measurements we can distinguish three different patterns reflecting cells variability within O-GCs group: (a) 39% cells showed small oscillations and [Ca(2+)](i) was 108±11nM; (b) 36% cells displayed yet small oscillations and [Ca(2+)](i) was 167±14nM; (c) 25% were cells with repetitive irregular oscillations that peaked until 2 fold basal value and [Ca(2+)](i) very variable, was 248±41nM. In O-GCs L-type VOCCs stimuli displayed different effects on [Ca(2+)](i) for both treatment in three basal patterns. In our study we demonstrated: (1) at resting the [Ca(2+)](i) is low (111±5nM) in M-GCs and tend to increasing in prehierarchical O-GCs; (2) L-type Ca(2+) channels are functionally expressed in the major part of O-GCs whereas they are not activated nor inhibited in M-GCs and in a percentage of O-GCs; (3) there are three different cellular types in prehierarchical O-GCs that may be associated with increasing stages of follicular development, based on their Ca(2+) pathway. Therefore, the functional response of L-type Ca(2+) channels in cultured laying hen prehierarchical GCs may be correlated with the functional maturation phase of laying hens ovarian. We hypothesize that the L-type Ca(2+)-dependent signaling could have a critical role in the regulatory mechanisms hormone mediated in hen ovarian cycle.     

Mechanotransduction through fibronectin-integrin focal adhesion in microvascular smooth muscle cells: is calcium essential?

It is believed that increased transmural pressure exerts force on vascular smooth muscle cells (VSMCs) and triggers Ca(2+) signaling as an initiating event responsible for the arteriolar myogenic response. However, the mechanisms linking the pressure increase to Ca(2+) signaling are unclear. We have shown previously using atomic force microscopy (AFM) that mechanical force induces a VSMC contractile response when applied to single fibronectin (FN; Sun Z, Martinez-Lemus LA, Hill MA, Meininger GA. Am J Physiol Cell Physiol 295; C268-C278, 2008) focal adhesion sites. This current study seeks to determine whether application of force to single focal adhesions can cause a change in VSMC Ca(2+). Experiments were performed in low passage (p3～10) as well as in freshly isolated skeletal muscle arteriole VSMCs. AFM-attached microbeads (5 μm) were coated with FN or collagen type I (CN-I) or type IV (CN-IV) and placed on a VSMC for 20 min, resulting in formation of a focal adhesion between the cell and the microbead. In low passage VSMCs, mechanically pulling on the FN-coated beads (800～3000 pN) did not induce a Ca(2+) increase but did cause a contractile response. In freshly isolated VSMCs, application of an FN or CN-I-coated bead onto the cell surface induced global Ca(2+) increases. However, these Ca(2+) increases were not correlated with the application of AFM pulling force to the bead or with the VSMC contractile responses to FN-coupled pulling. Chelating cytosolic Ca(2+) using BAPTA loading had no negative effect on the focal adhesion-related contractile response in both freshly isolated and low passage VSMCs, while the Rho-kinase inhibitor Y27632 abolished the micromyogenic response in both cases. These observations suggest that, in freshly isolated and cultured VSMCs, application of mechanical force to a focal adhesion does not invoke an acute global Ca(2+) increase. On the other hand, our data support a role for Rho-linked signaling mechanism involved in mechanotransduction leading to focal contraction that is independent of the need for a global increase in VSMC Ca(2+).     

Parathyroid hypertensive factor inhibits voltage-gated K+ channels in vascular smooth muscle cells

Parathyroid hypertensive factor (PHF) has been implicated in regulation of vascular smooth muscle tone and pathogenesis of several forms of hypertension. Earlier studies have suggested that PHF enhances the actions of other vasoconstrictors, while it has no in vitro vasoconstrictor property of its own. PHF was previously found to enhance the L-type Ca channel currents and intracellular Ca responses to depolarization in vascular smooth muscle cells (VSMCs). The present study examined whether PHF might act on K channels in the plasma membrane of VSMCs. Primary cultured VSMCs from rat tail artery were used. The whole-cell version of the patch-clamp technique was used under conditions in which there was no contribution of Ca-activated K channels to the outward current. Both purified and semipurified PHF inhibited the delayed rectifier type potassium current in a dose-dependent manner. The effect was time dependent and was first significantly different from the control current after 30 min. The inhibition of the delayed rectifier K channel was associated with a time-dependent decrease in the resting membrane potential. Therefore, PHF may alter VSMC cellular Ca responses by reducing the membrane potential to a level closer to the activation potential of Ca channels.     

L-type Ca(2+) channels, resting [Ca(2+)](i), and ET-1-induced responses in chronically hypoxic pulmonary myocytes

In the lung, chronic hypoxia (CH) causes pulmonary arterial smooth muscle cell (PASMC) depolarization, elevated endothelin-1 (ET-1), and vasoconstriction. We determined whether, during CH, depolarization-driven activation of L-type Ca(2+) channels contributes to 1) maintenance of resting intracellular Ca(2+) concentration ([Ca(2+)](i)), 2) increased [Ca(2+)](i) in response to ET-1 (10(-8) M), and 3) ET-1-induced contraction. Using indo 1 microfluorescence, we determined that resting [Ca(2+)](i) in PASMCs from intrapulmonary arteries of rats exposed to 10% O(2) for 21 days was 293.9 +/- 25.2 nM (vs. 153.6 +/- 28.7 nM in normoxia). Resting [Ca(2+)](i) was decreased after extracellular Ca(2+) removal but not with nifedipine (10(-6) M), an L-type Ca(2+) channel antagonist. After CH, the ET-1-induced increase in [Ca(2+)](i) was reduced and was abolished after extracellular Ca(2+) removal or nifedipine. Removal of extracellular Ca(2+) reduced ET-1-induced tension; however, nifedipine had only a slight effect. These data indicate that maintenance of resting [Ca(2+)](i) in PASMCs from chronically hypoxic rats does not require activation of L-type Ca(2+) channels and suggest that ET-1-induced contraction occurs by a mechanism primarily independent of changes in [Ca(2+)](i).     

Vascular smooth muscle cell membrane depolarization after NOS inhibition hypertension

Nitric oxide (NO) synthase (NOS) inhibition with N(omega)-nitro-L-arginine (L-NNA) produces L-NNA hypertensive rats (LHR), which exhibit increased sensitivity to voltage-dependent Ca(2+) channel-mediated vasoconstriction. We hypothesized that enhanced contractile responsiveness after NOS inhibition is mediated by depolarization of membrane potential (E(m)) through attenuated K(+) channel conductance. E(m) measurements demonstrated that LHR vascular smooth muscle cells (VSMCs) are depolarized in open, nonpressurized (-44.5 +/- 1.0 mV in control vs. -36.8 +/- 0.8 mV in LHR) and pressurized mesenteric artery segments (-41.8 +/- 1.0 mV in control vs. -32.6 +/- 1.4 mV in LHR). Endothelium removal or exogenous L-NNA depolarized control VSMCs but not LHR VSMCs. Superfused L-arginine hyperpolarized VSMCs from both the control and LHR groups and reversed L-NNA-induced depolarization (-44.5 +/- 1.0 vs. -45.8 +/- 2.1 mV). A Ca(2+)-activated K(+) channel agonist, NS-1619 (10 microM), hyperpolarized both groups of arteries to a similar extent (from -50.8 +/- 1.0 to -62.5 +/- 1.2 mV in control and from -43.7 +/- 1.1 to -55.6 +/- 1.2 mV in LHR), although E(m) was still different in the presence of NS-1619. In addition, superfused iberiotoxin (50 nM) depolarized both groups similarly. Increasing the extracellular K(+) concentration from 1.2 to 45 mM depolarized E(m), as predicted by the Goldman-Hodgkin-Katz equation. These data support the hypothesis that loss of NO activation of K(+) channels contributes to VSMC depolarization in L-NNA-induced hypertension without a change in the number of functional large conductance Ca(2+)-activated K(+) channels.     

Effects of Cl- channel blockers on endothelin-1-induced proliferation of rat vascular smooth muscle cells

The effects of Cl- channel blockers on endothelin-1 (ET-1)-induced proliferation of rat aortic vascular smooth muscle cells (VSMC) were examined. We found ET-1 concentration-dependently increased cell count and [3H]-thymidine incorporation into VSMC, with EC50 values of 24.8 and 11.4 nM, respectively. Both nifedipine and SK&F96365 inhibited 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC with the maximal inhibitory concentrations of 1 and 10 microM, respectively. DIDS inhibited 10 nM ET-1-induced increase in cell count and [3H]-thymidine incorporation into VSMC in a concentration-dependent manner, whereas other Cl- channel blockers including IAA-94, NPPB, DPC, SITS and furosemide did not produce these effects. 3 microM DIDS reduced 10 nM ET-1-induced sustained increase in cytoplasmic Ca2+ concentration ([Ca2+]) by 52%. Pretreatment of VSMC with 1 microM nifedipine completely inhibited the DIDS effect on 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC and sustained increase in [Ca2+]i, whereas pretreatment with 10 microM SK&F96365 did not completely block these effects of DIDS. DIDS did not affect ET-1-induced Ca2+ release and 30 mM KCl-induced increase in [Ca2+]i. Our data suggest that DIDS-sensitive Cl- channels mediate VSMC proliferation induced by ET-1 by mechanisms related to membrane depolarization and Ca2+ influx through voltage-dependent Ca2+ channels.     

Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.     

Positive heterotropic allosteric regulators of dihydropyridine binding increase the Ca2+ affinity of the L-type Ca2+ channel. Stereoselective reversal by the novel Ca2+ antagonist BM 20.1140.

The alpha 1-subunit of the voltage-dependent L-type Ca2+ channel has distinct, allosterically coupled binding domains for drugs from different chemical classes (dihydropyridines, benzothiazepines, phenylalkylamines, diphenylbutylpiperidines). (-)-BM 20.1140 (ethyl-2,2-di-phenyl-4-(1-pyrrolidino)-5-(2-picolyl)- oxyvalerate) is a novel Ca2+ channel blocker which potently stimulates dihydropyridine binding (K0.5 = 2.98 nM) to brain membranes. This property is shared by (+)-cis-diltiazem, (+)-tetrandrine, fostedil and trans-diclofurime, but (-)-BM 20.1140 does not bind in a competitive manner to the sites labeled by (+)-cis-[3H]diltiazem. (+)-cis-Diltiazem and (-)-BM 20.1140 have differential effects on the rate constants of dihydropyridine binding. (+)-BM 20.1140 reverses the stimulation of the positive allosteric regulators (pA2 value for reversal of (-)-BM 20.1140 stimulation = 7.4, slope 0.72). The underlying molecular mechanism of the potentiation of dihydropyridine binding has been clarified. The K0.5 for free Ca2+ to stabilize a high affinity binding domain for dihydropyridines on purified L-type channels from rabbit skeletal muscle is 300 nM. (+)-Tetrandine (10 microM) increases the affinity 8-fold (K0.5 for free Ca2+ = 30.1 nM) and (+)-BM 20.114 (10 microM) inhibits the affinity increase (K0.5 for free Ca2+ = 251 nM). Similar results were obtained with membrane-bound Ca(2+)-channels from brain tissue which have higher affinity for free Ca2+ (K0.5 for free Ca2+ = 132 nM) and for dihydropyridines compared with skeletal muscle. It is postulated that the dihydropyridine and Ca(2+)-binding sites are interdependent on the alpha 1-subunit, that the different positive heterotropic allosteric regulators (by their differential effects on Ca2+ rate constants) optimize coordination for Ca2+ in the channel pore and, in turn, increase affinity for the dihydropyridines.     

Neuropeptide W in the rat pancreas: potentiation of glucose-induced insulin release and Ca2+ influx through L-type Ca2+ channels in beta-cells and localization in islets

Neuropeptide W (NPW) is a regulatory peptide that acts via two subtypes of G protein-coupled receptors, GPR7 and GPR8. Evidence has been provided that NPW is involved in the central regulation of energy homeostasis and feeding behavior. In this study, we examined the effects of NPW on insulin release and localization of NPW in the rat pancreas. NPW (10-100 nM) significantly increased insulin release in the presence of 8.3 mM, but not 2.8 mM, glucose in the isolated rat islets. By fura-2 microfluorometry, NPW (1-100 nM) concentration-dependently increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) at 8.3 mM glucose in rat single beta-cells. The NPW-induced [Ca(2+)](i) increase was abolished under external Ca(2+)-free conditions and by an L-type Ca(2+) channel blocker nifedipine (10 microM). RT-PCR analysis revealed that mRNA for NPW was expressed in the rat pancreas and hypothalamus. Double immunohistochemical analysis showed that NPW-immunoreactivity was found in islets and co-localized with insulin-containing beta-cells, but not glucagon-containing alpha-cells and somatostatin-containing delta-cells. These results suggest that NPW could serve as a local modulator of glucose-induced insulin release in rat islets. NPW directly activates beta-cells to enhance Ca(2+) influx through voltage-dependent L-type Ca(2+) channels and potentiates glucose-induced insulin release.     

Large conductance Ca2+-activated K+ channels modulate endothelial cell outward currents and nitric oxide release in the intact rat superior mesenteric artery

Endothelial cells (EC) control vascular smooth muscle cell (VSMC) tone by release of paracrine factors. VSMC may also influence the EC layer, and therefore, the present study hypothesized that the opening of large-conductance Ca(2+) activated K(+) (BK(Ca)) channels may indirectly modulate EC hyperpolarization and nitric oxide (NO) release via myoendothelial gap junctions (MEGJ). To address this hypothesis 'in situ' EC ion current recordings, isolated VSMC patch clamp recordings, and simultaneous measurements of NO concentration and relaxation were conducted using segments of the rat superior mesenteric artery. In arteries constricted by α(1)-adrenoceptor activation, ACh (1 μM) evoked EC outward currents, vasorelaxation, and NO release. In contrast to preincubation with iberiotoxin (IbTx, 100nM) application of IbTx after ACh decreased EC outward currents, NO release and vasorelaxation. Furthermore, in phenylephrine (Phe)-contracted arteries treated with a gap junction uncoupler, cabenoxolone (CBX), IbTx failed to decrease ACh-evoked EC outward currents. In addition, CBX decreased EC outward currents, time constant of the capacitative transients, input capacitance, and increased input resistance. In isolated VSMC CBX did not affect BK(Ca) currents. Immunohistochemistry revealed only BK(Ca) channel positive staining in the VSMC layer. Therefore, the present results suggest that BK(Ca) channels are expressed in the VSMC, and that Phe by activation of VSMC BK(Ca) channels modulates ACh-evoked EC outward currents, NO release and vasorelaxation via MEGJ in rat superior mesenteric artery.     

Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain.     

Depolarization-induced Ca2+ release in ischemic spinal cord white matter involves L-type Ca2+ channel activation of ryanodine receptors

The mechanisms of Ca(2+) release from intracellular stores in CNS white matter remain undefined. In rat dorsal columns, electrophysiological recordings showed that in vitro ischemia caused severe injury, which persisted after removal of extracellular Ca(2+); Ca(2+) imaging confirmed that an axoplasmic Ca(2+) rise persisted in Ca(2+)-free perfusate. However, depletion of Ca(2+) stores or reduction of ischemic depolarization (low Na(+), TTX) were protective, but only in Ca(2+)-free bath. Ryanodine or blockers of L-type Ca(2+) channel voltage sensors (nimodipine, diltiazem, but not Cd(2+)) were also protective in zero Ca(2+), but their effects were not additive with ryanodine. Immunoprecipitation revealed an association between L-type Ca(2+) channels and RyRs, and immunohistochemistry confirmed colocalization of Ca(2+) channels and RyR clusters on axons. Similar to "excitation-contraction coupling" in skeletal muscle, these results indicate a functional coupling whereby depolarization sensed by L-type Ca(2+) channels activates RyRs, thus releasing damaging amounts of Ca(2+) under pathological conditions in white matter.