Functional impairment of renal afferent arteriolar voltage-gated calcium channels in rats with diabetes mellitus.

Experiments were performed to test the hypothesis that diabetes mellitus is associated with impaired afferent arteriolar responsiveness to opening of voltage-gated calcium channels. Diabetes was induced by injection of streptozocin (65 mg/kg, i.v.) and insulin was administered via an osmotic minipump to achieve moderate hyperglycemia. Sham rats received vehicle treatments. 2 wk later, the in vitro blood-perfused juxtamedullary nephron technique was used to allow videomicroscopic measurement of afferent arteriolar contractile responses to increasing bath concentrations of either Bay K 8644 or K+. Baseline afferent arteriolar diameter in kidneys from diabetic rats (26.4+/-1.2 microm) exceeded that of Sham rats (19.7+/-1.0 microm). Bay K 8644 evoked concentration-dependent reductions in afferent diameter in both groups of kidneys; however, arterioles from Sham rats responded to 1 nM Bay K 8644 while 100 nM Bay K 8644 was required to contract arterioles from diabetic rats. The EC50 for K+-induced reductions in afferent arteriolar diameter was greater in diabetic kidneys (40+/-4 mM) than in kidneys from Sham rats (28+/-4 mM; P < 0.05). In afferent arterioles isolated by microdissection from Sham rats and loaded with fura 2, increasing bath [K+] from 5 to 40 mM evoked a 98+/-12 nM increase in intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i responses to 40 mM K+ were suppressed in afferent arterioles from diabetic rats (delta = 63+/-5 nM), but were normalized by decreasing bath glucose concentration from 20 to 5 mM. These observations indicate that the early stage of insulin-dependent diabetes mellitus is associated with a functional defect in afferent arteriolar L-type calcium channels, an effect which may contribute to suppressed afferent arteriolar vasoconstrictor responsiveness and promote glomerular hyperfiltration.     

Direct contact between sympathetic neurons and rat cardiac myocytes in vitro increases expression of functional calcium channels.

To test the hypothesis that direct contact between sympathetic neurons and myocytes regulates expression and function of cardiac Ca channels, we prepared cultures of neonatal rat ventricular myocytes with and without sympathetic ganglia. Contractile properties of myocytes were assessed by an optical-video system. Contractility-pCa curves showed a 60% greater increase in contractility for innervated myocytes compared with control cells at 6.3 mM [Ca]0 (n = 8, P less than 0.05). Cells grown in medium conditioned by growth of ganglia and myocytes were indistinguishable physiologically from control cells. [Bay K 8644]-contractility curves revealed a 60 +/- 10% enhancement of the contractility response at 10(-6) M for innervated cells compared with control cells. The increased response to Bay K 8644 was not blocked by alpha- or beta-adrenergic antagonists. Moreover, increased efficacy of Bay K 8644 was maintained for at least 24 h after denervation produced by removal of ganglia from the culture. Dihydropyridine binding sites were assessed with the L channel-specific radioligand 3[H]PN200-110. PN200-110 binding sites were increased by innervation (51 +/- 5 to 108 +/- 20 fmol/mg protein, P less than 0.01), with no change in KD. Peak current-voltage curves were determined by whole-cell voltage clamp techniques for myocytes contacted by a neuron, control myocytes, and myocytes grown in conditioned medium. Current density of L-type Ca channels was significantly higher in innervated myocytes (10.5 +/- 0.4 pA/pF, n = 5) than in control myocytes (5.9 +/- 0.3 pA/pF, n = 8, P less than 0.01) or myocytes grown in conditioned medium (6.2 +/- 0.2 pA/pF, n = 10, P less than 0.01). Thus, physical contact between a sympathetic neuron and previously uninnervated neonatal rat ventricular myocytes increases expression of functional L-type calcium channels as judged by contractile responses to Ca0 and Bay K 8644, as well as by electrophysiological and radioligand binding properties.     

Angiotensin II amplifies arterial contractile response to norepinephrine without increasing Ca++ influx: role of protein kinase C.

We investigated whether the enhanced contractile response to norepinephrine caused by a subthreshold concentration of angiotensin II was associated with an increased 45Ca++ influx or net uptake. Rabbit facial artery segments were mounted isometrically to measure the 45Ca++ influx and net uptake in response to norepinephrine. The contractile response to norepinephrine (3 microM) in the presence of angiotensin II (0.1 nM) was 149.5 +/- 7.4% of control. This response amplification was not associated with changes in norepinephrine-induced 45Ca++ influx or net uptake. Angiotensin II also potentiated the contractile response to caffeine obtained in a Ca(++)-free buffer containing ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (2 mM) to 148.0 +/- 4.8% of control. In both cases, the amplification was prevented by pretreatment with either staurosporine (10 nM) or calphostin C (100 nM), two inhibitors of protein kinase C. We conclude that angiotensin II potentiation of norepinephrine-induced vascular tone occurs in the absence of changes in stimulated Ca++ entry. This potentiation may be due to an increase in intracellular sensitivity to Ca++, possibly mediated by protein kinase C.     

Endothelin-induced increases in vascular smooth muscle Ca2+ do not depend on dihydropyridine-sensitive Ca2+ channels.

Endothelin is a potent mammalian vasoconstrictive peptide with structural homology to cation channel-binding insect toxins. We tested the proposal that this peptide directly activates dihydropyridine-sensitive Ca2+ channels in cultured vascular smooth muscle (VSM) cells. First, we found that cell Ca2+ can be altered in VSM by activation of voltage-operated Ca2+ channels. KCl-induced depolarization and the dihydropyridine Ca2+ channel agonist (-) Bay K 8644 (10 microM) both raised cell Ca2+ more than twofold; the effect of KCl was blocked by the inhibitory enantiomer, (+) Bay K 8644 (40 microM). Similar responses were observed in Chinese hamster ovary (CHO) cells. Synthetic endothelin (4 x 10(-8) M) raised Ca2+ in VSM but not CHO cells from 100 +/- 17 to 561 +/- 34 nM within 12 s. Ca2+ subsequently fell to basal levels after 30 min. Half maximal Ca2+ response was at 4 x 10(-9) M endothelin. Unlike endothelin, thrombin raised Ca2+ in both VSM and CHO cells. The Ca2+ responses to endothelin and thrombin were not affected by nicardipine (1 microM), (+) Bay K 8644, or Ca2+-free solutions. Lastly, both hormones caused release of inositol phosphates in VSM cells. However, the response to thrombin was more than 10-fold larger and was more rapid than the response to endothelin; the thrombin response was sensitive to pertussis toxin, while the response to endothelin was not. Thus endothelin, like thrombin, raises cell Ca2+ in VSM by mobilization of intracellular stores and not by activation of dihydropyridine-sensitive Ca2+ channels. However, their receptors are distinct and they exhibit important differences in signal transduction.     

Responses of arterial smooth muscle from normotensive and pre-eclamptic subjects to the calcium channel agonist,Bay K 8644

The effect of Bay K 8644, a dihydropyridine Ca2+ agonist, on in vitro contractile responses of inferior epigastric arteries from normotensive (N) and pre-eclamptic (P) subjects has been investigated, with a view to further defining the mechanism of the increased vascular sensitivity associated with pregnancy-induced hypertension. Bay K 8644 (10(-10)-10(-7) M) caused dose-dependent contractions of N as well as P arteries under resting conditions in the order: P greater than N and caused development of rhythmic contractions in both N and P arteries. Bay K 8644 effects were prevented by 3 X 10(-8) M Nifedipine (a Ca+2 antagonist). Bay K 8644 also significantly (P less than 0.05) enhanced the sensitivity as well as maximal contractile responses to CaCl2 in 40 mM K+-depolarized Ca-depleted N and P arteries in the order: P greater than N. The results suggest that the increased peripheral vascular sensitivity associated with pregnancy-induced hypertension may be due, at least in part, to enhanced activity of the potential-sensitive Ca2+ channels in arterial smooth muscle plasmalemma.     

Contractile effects of Bay k 8644, a dihydropyridine calcium agonist, on isolated femoral arteries from spontaneously hypertensive rats

Agonist actions of methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate (Bay k 8644) were investigated in femoral and mesenteric arteries from 6-week-old spontaneously hypertensive rats (SHRs), and data compared with findings in normotensive Wistar-Kyoto rats (WKYs). The addition of Bay k 8644 produced a dose-dependent contraction in SHR femoral artery with a pD2 value of 8.55. Maximum contraction induced by this agonist (1 X 10(-7) M) was comparable to the maximum developed by K+-depolarization. Bay k 8644 was much less effective in eliciting the contractile responses on WKY femoral artery. Contractile responses of mesenteric and tail arteries to Bay k 8644 were weak and were not significantly different between SHR and WKY. Thoracic aorta was sensitive to the contractile response to Bay k 8644, but the sensitivity was not significantly different between SHR and WKY. Increased responsiveness to exogenously applied K+ was also observed in SHR femoral artery as compared to WKY. Contractile responses of SHR femoral artery to Bay k 8644 were antagonized competitively by nifedipine (pA2 = 8.36), a dihydropyridine Ca++ antagonist, but noncompetitively by diltiazem, a non-dihydropyridine Ca++ antagonist. When the effect of nifedipine on the dose-response curve for Bay k 8644 was determined in WKY femoral artery, there was a similar extent of rightward displacement of the dose-response curve to that seen in SHR. Nifedipine was less efficacious in relaxing the contractile response to Bay k 8644 compared to the contractile response to K+ in SHRs femoral arteries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in cytosolic calcium level in vascular smooth muscle strip measured simultaneously with contraction using fluorescent calcium indicator fura 2

In rat aortic strips, muscle contraction was recorded simultaneously with cytosolic Ca++ level, which was indicated by the 500 nm fluorescence of Ca++ indicator, fura 2, due to excitation at either 340 nm (F340) or 380 nm (F380) and the ratio of F340 to F380 (R340/380). On the addition of 72.7 nM K+ or 1 microM norepinephrine, muscle contraction followed the increase in R340/380 (resulted from the increased F340 and the decreased F380). Cytosolic Ca++ concentrations of resting, 72.7 mM K+-stimulated and 1 microM norepinephrine-stimulated aortas were tentatively calculated as 228 +/- 25, 1784 +/- 154 and 1528 +/- 180 nM, respectively. Cumulative addition of K+ or norepinephrine induced concentration-dependent increase in both muscle tension and R340/380. However, norepinephrine induced greater contraction than K+ when both of these stimulants induced similar increase in R340/380. Addition of 10 mM tetraethylammonium and 1 microM Bay k8644 caused rhythmic contractions which followed the rhythmic changes in R340/380. EGTA decreased the muscle contraction and decreased R340/380. In Ca++-free solution, addition of 10 microM norepinephrine or 20 mM caffeine induced transient increase in both muscle tension and R340/380. Tension changes always were preceded by the fluorescent changes. Verapamil (10 microM) decreased both tension development and R340/380 in high K+- and norepinephrine-stimulated tissues. Sodium nitroprusside (1 microM) also decreased both tension and R340/380 in norepinephrine-stimulated tissues, whereas it decreased tension more strongly than R340/380 in high K+-stimulated tissues. These results indicate that vasoconstrictors and vasodilators may modulate smooth muscle contraction by changing the cytosolic Ca++ concentrations and also by changing the sensitivity of contractile elements to Ca++.     

Effects of Bay k 8644 and nifedipine on isolated dog cerebral, coronary and mesenteric arteries

Vasoconstrictor effects of Bay k 8644, a dihydropyridine Ca++ agonist, and vasorelaxant effects of nifedipine were investigated in helical strips of dog cerebral (basilar, posterior cerebral and middle cerebral) and peripheral (coronary and mesenteric) arteries. The addition of Bay k 8644 produced a dose-dependent contraction in the absence of any contractile agent in the basilar artery with a pD2 value of 8.53. Similar sensitivity to Bay k 8644 was observed in the posterior cerebral, middle cerebral or coronary artery. Bay k 8644 was much less effective in producing a contraction in the mesenteric artery. An elevation of the concentration of extracellular K+ eliminated the difference between the responses to Bay k 8644 in the basilar and mesenteric artery. Contractile responses of the basilar artery to Bay k 8644 were antagonized competitively by nifedipine (pA2 = 8.17), but non-competitively by diltiazem. The pA2 values for nifedipine antagonism of Bay k 8644 responses with the elevated K+ were the same between the basilar and mesenteric arteries. Increased sensitivity to exogenously added K+ also was observed in cerebral and coronary arteries when compared with the mesenteric artery. The addition of nifedipine to an unstimulated strip produced a dose-dependent relaxation in cerebral and coronary arteries, but not in the mesenteric artery. When the cerebral and peripheral arteries were contracted with K+ to the same magnitude, nifedipine produced similar relaxations among these arteries. Nifedipine was less efficacious in antagonizing the contractile response to Bay k 8644 compared with the contractile response to K+ in cerebral arteries. These results suggest that 1) the voltage-dependent Ca++ channels in the cerebral and coronary arteries are in different states of activation from those in the mesenteric artery, 2) Bay k 8644 contracts the cerebral and coronary arteries by acting primarily on the same site, presumably dihydropyridine receptors of the voltage-dependent Ca++ channels at which nifedipine acts, 3) the dihydropyridine receptors were the same between the basilar and mesenteric arteries and 4) there may be a difference in the state of the Ca++ channel in the arteries between the stimulation with Bay k 8644 and K+-depolarization.     

Competitive and cooperative effects of Bay K8644 on the L-type calcium channel current inhibition by calcium channel antagonists

Phenylalkylamines, benzothiazepines, and dihydropyridines bind noncompetitively to the L-type calcium channel. The molecular mechanisms of this interaction were investigated in enzymatically isolated rat ventricular myocytes using the whole-cell patch-clamp technique. When applied alone, felodipine, verapamil, and diltiazem inhibited the L-type calcium current with values of inhibitory constant (K(B)) of 11, 246, and 512 nM, respectively, whereas 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K8644) activated I(Ca) with activation constant (K(A)) of 33 nM. Maximal activation of I(Ca) by 300 nM Bay K8644 strongly reduced the inhibitory potency of felodipine (apparent K(B) of 165 nM), significantly reduced the inhibitory potency of verapamil (apparent K(B) of 737 nM), but significantly increased the inhibitory potency of diltiazem (apparent K(B) of 310 nM). In terms of a new pseudoequilibrium two-drug binding model, the interaction between the dihydropyridine agonist Bay K8644 and the antagonist felodipine was found purely competitive. The interaction between Bay K8644 and verapamil or diltiazem was found noncompetitive, and it could be described only by inclusion of a negative interaction factor nu = -0.60 for verapamil and a positive interaction factor nu = +0.24 for diltiazem. These results suggest that at physiological membrane potentials, the L-type calcium channel cannot be simultaneously occupied by a dihydropyridine agonist and antagonist, whereas it can simultaneously bind a dihydropyridine agonist and a nondihydropyridine antagonist. Generally, the effects of the drugs on the L-type calcium channel support a concept of a channel domain responsible for binding of calcium channel antagonists and agonists changing dynamically with the membrane voltage and occupancy of individual binding sites.     

Agonist actions of Bay K 8644, a dihydropyridine derivative, on the voltage-dependent calcium influx in smooth muscle cells of the rabbit mesenteric artery

Actions of methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2- trifluoromethylphenyl)-pyridine-5-carboxylate (Bay K 8644) on the mechanical response evoked in intact and skinned mesenteric artery of the rabbit were investigated. The data were compared to that of nisoldipine, another dihydropyridine derivative Bay K 8644 increased the amplitudes of both the phasic and tonic components of the K+-induced contraction which is due to an increase in the voltage-dependent influx of Ca ion. Bay K 8644 antagonized competitively the actions of nisoldipine (a Ca antagonist) on the tonic but not on the phasic component of the K+-induced contraction. The contractions caused by high concentrations of norepinephrine were enhanced to a greater extent by Bay K 8644 than that evoked by lower concentrations of norepinephrine. Bay K 8644 had no effect on Ca++ extrusion from cells, which was estimated from the change in amplitudes of the norepinephrine-induced contractions in Na+- and Ca++-free solutions. This agent had no effect on the contractile proteins and Ca storage sites, as estimated from the Ca++- or caffeine-induced contraction observed in skinned muscles. The results suggested that Bay K 8644 acts primarily on the voltage-dependent Ca++ channel, presumably the same site at which other dihydropyridine derivatives (Ca antagonists) act, and that the influx of Ca++ is accelerated.     

Vascular and cardiac effects of a new dihydropyridine derivative, YC-170: a comparison with Bay K 8644

The pharmacological effects of YC-170, a new dihydropyridine derivative, were studied in the rabbit aortic strips and guinea pig cardiac preparations and compared with those of Bay K 8644. In the rabbit aortic strips, YC-170 produced contraction in normal physiological saline solution ([K+]0 = 5.9 mM) in a concentration-dependent manner. Increasing the [K+]0 of the medium to 15 mM enhanced the contractile response. The maximum contraction produced by YC-170 at [K+]0 of 15 mM was comparable to that by Bay K 8644. However, YC-170 induced relaxation when the strip was contracted by 60 mM K+. In guinea pig left atrium, YC-170 produced a positive inotropic effect in a concentration-dependent manner, but its extent was far less than that of Bay K 8644. Like Bay K 8644, however, YC-170 increased the time to peak tension and relaxation time of the isometric tension, and prolonged the action potential duration. YC-170 failed to produce a positive inotropic action in the papillary muscle in which Bay K 8644 was a potent positive inotropic agent. In spontaneously beating right atria, YC-170 caused a negative chronotropic effect, whereas Bay K 8644 a positive one. The positive inotropic and vasoconstrictor effects of YC-170 were antagonized competitively by a Ca++ antagonist nicardipine. When the left atria were depolarized with high-K+ medium, the positive inotropic effect of YC-170 was attenuated progressively with increasing [K+]0 and at 13.2 mM K+ a negative inotropic effect was induced by YC-170.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium dependency and free calcium concentrations during insulin secretion in a hamster beta cell line.

Using a glucose-responsive beta cell line, we tested the hypothesis that the free cytosolic Ca2+ concentration ([Ca2+]i) is the primary signal that couples a stimulus to insulin secretion, and examined the involvement of the extracellular Ca2+ pool in this process. Glucose or depolarization of the beta cell with 40 mM K+ stimulated a monophasic release of insulin directly proportional to the extracellular Ca2+ concentration. 40 mM K+ increased 45Ca2+ uptake and increased [Ca2+]i, which was measured with quin 2, 4.7-fold, from 56 +/- 3 to 238 +/- 17 nM. With high glucose, 45Ca2+ uptake did not increase, and [Ca2+]i was unchanged or fell slightly. There was a striking correlation between inhibitory effects of verapamil, the Ca2+ channel blocker, on insulin secretion and the rise in [Ca2+]i evoked by K+. Higher concentrations of verapamil were required to inhibit glucose- than K+-stimulated insulin secretion (dose giving half-maximal effect of 1.4 X 10(-4) M vs. 6.0 X 10(-7) M). Incubation in Ca2+-free, 1 mM EGTA buffer for 30 min lowered [Ca2+]i to 14 +/- 2 nM, and inhibited acute insulin release to both secretagogues. If high glucose was present in the Ca2+-free period, reintroduction of 2.5 mM Ca2+ in high glucose restored insulin secretion only to the basal rate. However, if low glucose was present during the Ca2+-free period, high glucose and 2.5 mM Ca2+ triggered a full first-phase insulin response. These data suggest that high glucose generates a non-Ca2+ signal that turns over rapidly and provide direct evidence that K+ triggers insulin release by drawing extracellular Ca2+ into the beta cell through verapamil-sensitive Ca2+ channels. However, an increase [Ca2+]i is not the primary signal that evokes glucose-stimulated insulin release in this beta cell line.     

Interactions between phorbol esters and agents which increase cytosolic calcium in the guinea pig parenchymal strip: direct and indirect effects on the contractile response

In the guinea pig parenchymal strip the cumulative concentration-response curve to 4 beta-phorbol dibutyrate (4 beta-PDBu) occurred over the range 10 nM to 10 microM, the response to 10 microM being 246 +/- 22% of the mean maximum histamine response. A "cocktail" of agents which inhibit synthesis and/or release of mediators (mepyramine, 1 microM; indomethacin, 1 microM; mepacrine, 300 microM; atropine, 10 microM; cimetidine, 10 microM; phentolamine, 10 microM; and quercetin, 50 microM) had no effect on the cumulative 4 beta-PDBu concentration-response curve, indicating that the response was not due to the release of noradrenaline, acetylcholine, histamine, platelet activating factor or eicosanoids. When the cumulative curves to vanadate and A23187 were measured in the presence of the cocktail of inhibitors, the vanadate response was unaffected, but the response to A23187 was reduced marginally. Threshold concentrations of four agents which increase cytosolic calcium (Bay K 8644, 1 and 10 nM; ionomycin, 100 nM; A23187, 100 nM; and vanadate, 3 microM) shifted the cumulative 4 beta-PDBu curve to the left. A threshold concentration of 4 beta-PDBu (30 nM) shifted the cumulative-response curve of vanadate to the left, had no effect on the Bay K 8644 response and left-shifted the A23187 curve whether the tissue was immersed in cocktail-containing Krebs' solution or not. Threshold concentrations of A23187 and 4 beta-PDBu added together in the inhibitory cocktail caused only a transient contraction as compared to a sustained contraction with 1 microM 4 beta-PDBu.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iberiotoxin-induced block of Ca2+-activated K+ channels induces dihydropyridine sensitivity of ACh release from mammalian motor nerve terminals

The role which Ca(2+)-activated K(+) (K(Ca)) channels play in regulating acetylcholine (ACh) release was examined at mouse motor nerve terminals. In particular, the ability of the antagonist iberiotoxin to recruit normally silent L-type Ca(2+) channels to participate in nerve-evoked release was examined using conventional intracellular electrophysiological techniques. Incubation of cut hemidiaphragm preparations with 10 microM nimodipine, a dihydropyridine L-type Ca(2+) channel antagonist, had no significant effect on quantal content of end-plate potentials. Nevertheless, 1 microM S-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K 8644) enhanced quantal content to 134.7 +/- 3.5% of control. Iberiotoxin (150 nM) increased quantal content to 177.5 +/- 9.9% of control, whereas iberiotoxin plus nimodipine increased quantal content to only 145.7 +/- 10.4% of control. Coapplication of 1 microM Bay K 8644 with iberiotoxin did not significantly increase quantal content further than did treatment with iberiotoxin alone. The effects of iberiotoxin and nimodipine alone or in combination on the miniature end-plate potential (MEPP) frequency following KCl-induced depolarization were examined using uncut hemidiaphragm preparations. Nimodipine alone had no effect on MEPP frequency from preparations incubated in physiological saline containing 5 to 20 mM KCl. Moreover, iberiotoxin alone or combined with nimodipine also had no effect on MEPP frequency in physiological salines containing 5 to 15 mM KCl. At 20 mM KCl, however, iberiotoxin significantly increased MEPP frequency to 125.6% of iberiotoxin-free values; combined treatment with nimodipine and iberiotoxin prevented this increase in MEPP frequency. Thus, loss of functional K(Ca) channels unmasks normally silent L-type Ca(2+) channels to participate in ACh release from motor nerve terminals, particularly under conditions of intense nerve terminal depolarization.     

Sustained contraction of vascular smooth muscle: calcium influx or C-kinase activation?

We have investigated the relative contributions of Ca++ influx and C-kinase activation to the sustained contraction of smooth muscle of rabbit aorta. In physiological salt solution (PSS), the alpha adrenergic agonist, phenylephrine (PhE), induced a rapid initial contraction followed by a maintained tonic contraction whereas the C-kinase activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused only a slow tonic contractile response. Both PhE- and TPA-induced contractions were accompanied by a significant increase in the unidirectional 45Ca influx. The tonic phase of PhE contraction and the slow contractile response of TPA also were reduced, but not abolished completely in Ca++-free solution containing 2 mM ethylene glycol bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid. In addition, the relatively specific C-kinase inhibitor, H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine], reversibly inhibited the TPA-induced contraction in PSS and almost abolished the TPA response in Ca++-free solution. On the other hand, H-7 caused only partial inhibition (30.2% +/- 4.09, n = 5) of the PhE sustained contraction in PSS and abolished completely the residual PhE maintained response in Ca++-free solution. The H-7 inhibition of the PhE sustained contraction was reversible in both PSS and Ca++-free solution. Furthermore, TPA alone could not maintain the contractile response initiated by a high K+ depolarizing solution upon replacement of the high K+ solution by normal PSS. These findings emphasize the importance of Ca++ influx and suggest only a minor role of C-kinase in maintaining the tonic contraction of vascular smooth muscle.     

Calcium channels involved in K+- and veratridine-induced increase of cytosolic calcium concentration in human cerebral cortical synaptosomes.

Human cerebral cortical synaptosomes were used to study voltage-dependent Ca(2+) channels mediating calcium influx in human axon terminals. Synaptosomes were depolarized by elevation of the extracellular K(+) concentration by 30 mM or by the addition of veratridine (10 microM). Increase in cytosolic concentration of calcium [Ca(2+)](i) induced by either stimulus was abolished in the absence of extracellular Ca(2+) ions. omega-Agatoxin IVA inhibited the K(+)-induced [Ca(2+)](i) increase concentration-dependently (IC(50): 113 nM). omega-Conotoxin GVIA (0.1 microM) inhibited K(+)-induced [Ca(2+)](i) increase by 20%. omega-Conotoxin MVIIC (0.2 microM) caused an inhibition by 85%. Nifedipine (1 microM) had no effect on K(+)-induced [Ca(2+)](i) increase. Veratridine-induced increase in [Ca(2+)](i) was inhibited by omega-conotoxin GVIA (0.1 microM) and omega-Agatoxin IVA (0.2 microM; by about 25 and 45%, respectively). Nifedipine inhibited the veratridine-evoked [Ca(2+)](i) increase concentration-dependently (IC(50): 4.9 nM); Bay K 8644 (3 microM) shifted the nifedipine concentration-response curve to the right. Mibefradil (10 microM) abolished the increase in [Ca(2+)](i) evoked by K(+) and reduced the increase evoked by veratridine by almost 90%. KB-R7943 (3 microM) an inhibitor of the Na(+)/Ca(2+) exchanger NCX1, decreased the increase in [Ca(2+)](i) evoked by veratridine by approximately 20%. It is concluded that the increase in [Ca(2+)](i) after K(+) depolarization caused by Ca(2+) influx predominantly via P/Q-type Ca(2+) channels and after veratridine depolarization via N- and P/Q-type, but also by L-type Ca(2+) channels. The toxin- and nifedipine-resistant fraction of the veratridine response may result both from influx via R-type Ca(2+) channels and by Ca(2+) inward transport via Na(+)/Ca(2+) exchanger.     

Ca++ inhibition of isoproterenol responses in mammalian skeletal muscle

In noncontracting mouse hemidiaphragms incubated in modified Krebs-Ringer--bicarbonate buffer with 10 mM Ca++, isoproterenol-stimulated phosphorylase a formation, conversion of phosphorylase kinase to the activated form, elevation of cyclic AMP-dependent protein kinase activity ratios and increase in cyclic AMP concentrations were reduced 35 to 50% over the responses in buffer with 2.5 mM Ca++. In buffer with 10 mM Ca++, the initial rate of isoproterenol-stimulated cyclic AMP accumulation was 59% of that in buffer with 2.5 mM Ca++. The inhibitory action of Ca++ on cyclic AMP accumulation was antagonized by verapamil, but not by inhibitors of cyclic nucleotide phosphodiesterase activity. In buffer with 2.5 mM Ca++, isoproterenol-stimulated cyclic AMP accumulation was inhibited by A23187 and caffeine, agents that can increase intracellular Ca++ concentrations. In addition to Ca++, high concentrations of Co++, Ni++, Mn++ and, to a lesser extent, Sr++ inhibited the isoproterenol response. The results of these studies indicate that high buffer Ca++ concentrations inhibit the response of the glycogenolytic pathway to isoproterenol by an action on cyclic AMP formation. We propose that the site of the inhibitory action of Ca++ is the divalent metal activator site associated with hormone-stimulated adenylate cyclase activity.     

Dihydropyridine-sensitive and -insensitive components of acetylcholine release from rat motor nerve terminals

Effects of the dihydropyridine (DHP) calcium channel agonist Bay K 8644 on spontaneous and neurally evoked release of acetylcholine were measured using conventional intracellular microelectrode recording techniques at rat neuromuscular junctions of preparations that were transected to prevent contraction ("cut muscle preparation"). At concentrations of 0.65 to 2 microM Bay K 8644 caused significant increases in end-plate potential amplitude and mean quantal content in cut muscle preparations, but no effect in uncut preparations in which contractions were blocked by using d-tubocurarine (1 microM). The dose-dependence of this effect occurred over a very narrow concentration range. This increase in quantal content, which occurred within 5 to 10 min of application of Bay K 8644, could be blocked by pretreatment or reversed by subsequent treatment of the preparation with nimodipine, a DHP antagonist. Nimodipine itself had no effect on quantal content. At concentrations of Bay K 8644 in excess of 1 microM, increase quantal content was usually followed by a subsequent complete failure of nerve-evoked release of transmitter. Administration of Bay K 8644 was also associated with an increase in the frequency of miniature end-plate potentials (MEPPs). This effect was observed in 5 of 6 "cut" and only 1 of 6 "uncut" preparations. Increase of MEPP frequency occurred after a latent period of 15 to 25 min of treatment with Bay K 8644, and was not prevented pretreatment with nimodipine. Nimodipine itself had no effect on MEPP frequency. Increased MEPP frequency occurred in cut preparations treated with Bay K 8644, but with solutions to which no extracellular Ca++ was added.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nifedipine and Bay K 8644 Induce an increase of

BACKGROUND: The dihydropyridine-induced vasorelaxation is partly dependent on the endothelium, which does not express L-type calcium channels. Because nitric oxide (NO) is one of the most important endothelium-derived vasorelaxing factors, we investigated how the calcium antagonist nifedipine and the calcium agonist Bay K 8644 modulate intracellular calcium and NO formation in porcine endothelial cells. METHODS AND RESULTS: NO formation of porcine aortic endothelial cell cultures and of native endothelium of intact porcine coronary arteries was measured with an electrochemical electrode, and the intracellular concentration of Ca(2+) [Ca(2+)](i) was evaluated using the Fura-2 technique. Nifedipine induced a concentration-dependent [0,01-1 μmol/L] increase in [Ca(2+)](i) and NO formation in cultured porcine aortic endothelial cells, and moreover a dose-dependent NO formation in native endothelial cells from intact porcine coronary arteires, which was higher than in cultured cells. This effect was inhibited by N-nitro-l-arginine, a specific NO synthase inhibitor. Bay K 8644 caused a [Ca(2+)](i) increase and NO release in cultured cells, too, although to a lesser extent. Nifedipine-induced and Bay K 8644-induced [Ca(2+)](i) rise could be blocked by removal of extracellular calcium, indicating that a calcium influx may be involved. CONCLUSIONS: The calcium antagonist nifedipine as well as the calcium agonist Bay K 8644 cause an increase of [Ca(2+)](i) and NO in porcine endothelium. Therefore, these effects seem to be related to the dihydropyridines as a substance class, which may explain the endothelial component in dihydropyridine-induced vasorelaxation.     

Binding of the dihydropyridine calcium channel blocker (+)-[3H] isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-5-methoxycarbonyl-2, 6-dimethyl-3-pyridinecarboxylate (PN200-110) to RINm5F membranes and cells: characterization and functional significance.

This report provides direct evidence for a dihydropyridine receptor/calcium channel in the insulin-secreting beta-cell line RINm5F. The receptor/channel can modulate the intracellular Ca++ concentration and the resultant insulin secretion by regulating the influx of extracellular Ca++ through dihydropyridine-sensitive voltage-dependent L-type Ca++ channels. Elevated extracellular K+ or the dihydropyridine Ca++ channel agonist, BAY k 8644 [methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethyl- phenyl)pyridine-5-carboxylate], stimulated the uptake of 45Ca++, raised [Ca++]i, and increased insulin secretion in a concentration-dependent manner. These actions were inhibited by L-type Ca++ channel blockers including nitrendipine, verapamil and diltiazem. (+)-[3H]PN200-110 bound specifically with high affinity to RINm5F cell membranes (Kd approximately 200 pM). Specific binding was inhibited competitively by dihydropyridines whereas phenylalkylamines inhibited incompletely (+)-[3H]PN200-110 binding, consistent with an allosteric interaction. The benzothiazepine diltiazem had no effect on (+)-[3H]PN200-110 binding in the presence of Ca++, but increased binding allosterically in the absence of Ca++ (in the presence of EGTA). Maximal (+)-[3H]PN200-110 binding required divalent cations, with Mg++, Mn++ and Ba++ essentially as effective as Ca++ in reversing the effects of EGTA, whereas binding was not supported by Cd++ or La . Specific high affinity (+)-[3H]PN200-110 binding was also demonstrated in intact RINm5F cells and shown to be modulated by membrane potential. Depolarization of the cells by raising extracellular K+ from 5 to 80 mM increased the affinity of (+)-[3H]PN200-110 4- to 5-fold (decreased Kd) with no significant effect on the maximum number of binding sites.