The effect of dihydropyridine calcium agonists and antagonists on neuronal voltage sensitive calcium channels

The effect of dihydropyridine agonists and antagonists on neuronal voltage sensitive calcium channels was investigated. The resting intracellular calcium concentration of synaptosomes prepared from whole brain was 110 +/- 9 nM, as assayed by the indicator quin 2. Depolarisation of the synaptosomes with K+ produced an immediate increase in [Ca2+]i. The calcium agonist Bay K 8644 and antagonist nifedipine did not affect [Ca2+]i under resting or depolarising conditions. In addition, K+ stimulated 45Ca2+ uptake into synaptosomes prepared from the hippocampus was insensitive to Bay K 8644 and PY 108-068 in normal or Na+ free conditions. In neuronally derived NG108-15 cells the enantiomers of the dihydropyridine derivative 202-791 showed opposite effects in modulating K+ stimulated 45Ca2+ uptake. (-)-R-202-791 inhibited K+ induced 45Ca2+ uptake with an IC50 of 100 nM and (+)-S-202-791 enhanced K+ stimulated uptake with an EC50 of 80 nM. These results suggest that synaptosomal voltage sensitive calcium channels either are of a different type to those found in peripheral tissues and cells of neural origin or that expression of functional effects of dihydropyridines requires different experimental conditions to those used here.     

Profile of the oppositely acting enantiomers of the dihydropyridine 202-791 in cardiac preparations: receptor binding, electrophysiological, and pharmacological studies

Receptor binding, electrophysiological, and inotropic effects of the pure dihydropyridine enantiomers (+)S202-791 and (-)R202-791 were studied in cardiac preparations. The KI for (+)S202-791 binding correlated with the ED50's for an increase in contractile force and an increase in calcium current, the latter effect occurring at depolarized as well as resting holding potentials. The KI for (-)R202-791 binding was much lower than the IC50's for inhibition of calcium current measured at holding potentials of -80 or -90 mV and a negative inotropic effect, but correlated closely with the IC50 for inhibition of calcium current measured at -30 mV. Thus, (+)S202-791, is a voltage independent calcium channel activator and (-)R202-791 is a voltage dependent calcium channel inhibitor.     

Modulation of depolarization stimulated 45Ca2+ uptake in cultured neuronal cells by calcium channel activators and antagonists

The effect of dihydropyridine calcium agonists and antagonists on 45Ca2+ uptake into primary neuronal cell cultures was investigated. K+ stimulated neuronal 45Ca2+ accumulation in a concentration dependent manner. This effect was further enhanced by the calcium agonists Bay K 8644 and (+)-(S)-202-791 with EC50 values of 21 nM and 67 nM respectively. The calcium antagonists PN 200-110 and (-)-(R)-202-791 inhibited Bay K 8644 (1 microM) stimulated uptake with IC50 values of 20 nM and 130 nM respectively. 45Ca2+ efflux from neuronal cells was measured in the presence and absence of Na+. Efflux occurred at a much greater rate from cells incubated in the presence of Na+, indicating the existence of an active Na+/Ca2+ exchanger in these neurons. The data suggests that voltage sensitive calcium channels of these neurons are sensitive to dihydropyridines and thus that dihydropyridine binding sites have a functional role in these neuronal cultures.     

Calcium channel agonist and antagonist effects of the stereoisomers of the dihydropyridine 202-791

The effects of the pure stereoisomers of the novel dihydropyridine 202-791 on voltage sensitive calcium channels in nerve and cardiac muscle were examined. The (-)-isomer blocked depolarization-induced uptake of 45Ca2+ into NG108-15 neuroblastoma X glioma cells, blocked the depolarization-induced release of [3H]-norepinephrine from PC12 cells and reduced the Vmax of the slow response action potential recorded from guinea pig papillary muscle. In contrast, the (+)-isomer enhanced these same processes. In papillary muscle, greater enhancement of the slow responses was observed at lower stimulation frequencies. Thus, the (-) and (+) stereoisomers of 202-791 can be shown to be calcium channel antagonist and agonist respectively.     

Calcium uptake studies of 1,4-dihydropyridine agonists into rabbit aortic smooth muscle cells in culture

The effects of the three dihydropyridine calcium channel agonists (+/-)BAY K 8644, (+)202-791 and (+/-)CGP 28392 on 45Ca++ uptake were studied in cultures of rabbit aortic smooth muscle cells. At 10(-7) M each agonist enhanced 45Ca++ uptake in 15-50 mM K+ but had no effect on the basal 45Ca++ uptake at 5 mM K+. At the uptake threshold of 15 mM K+ each agonist potentiated 45Ca++ uptake in a dose-dependent manner with half maximal effects at 2.4 nM for (+/-)BAY K 8644, 22 nM for (+)202-791 and 18 nM for (+/-)CGP 28392. The agonists showed no significant antagonistic activity. Responses were antagonized competitively by nifedipine and non-competitively by (+/-)D-600. The 45Ca++ uptake dose-response curves and the half maximal effects of the three agonists were over the same range of concentrations as their inhibition of [3H]nitrendipine binding to rat ventricular receptor membrane preparations. The data suggest that these cells mimic the calcium uptake by the intact aorta better than commercial vascular smooth muscle lines or cardiac cells.     

Effects of Ca2+-channel antagonists on nucleoside and nucleobase transport in human erythrocytes and cultured mammalian cells

Lidoflazine strongly inhibited the equilibrium exchange of uridine in human erythrocytes (Ki approximately 16 nM). Uridine zero-trans influx was similarly inhibited by lidoflazine in cultured HeLa cells (IC50 approximately to 80 nM), whereas P388 mouse leukemia and Novikoff rat hepatoma cells were three orders of magnitude more resistant (IC50 greater than 50 microM). Uridine transport was also inhibited by nifedipine, verapamil, diltiazem, prenylamine and trifluoperazine, but only at similarly high concentrations in both human erythrocytes and the cell lines. IC50 values ranged from about 10 microM for nifedipine and about 20 microM for verapamil to more than 100 microM for diltiazem, prenylamine and trifluoperazine. The concentrations required for inhibition of nucleoside transport are several orders higher than those blocking Ca2+ channels. Lidoflazine competitively inhibited the binding of nitrobenzylthioinosine to high-affinity sites in human erythrocytes, but did not inhibit the dissociation of nitrobenzylthioinosine from these sites on the transporter as is observed with dipyridamole and dilazep.     

The effect of organic calcium channel modulators on the germination ofVaucheria longicaulis aplanospores

Summary InVaucheria longicaulis var.macounii aplanospore germination and filament growth are severely inhibited by the Ca²⁺-channel antagonists (–)202–791, diltiazem, nifedipine and verapamil, whereas the agonists (+)202–791, Bay K-8644 and CGP-28392 stimulate those processes. Both antagonist and agonist actions suggest that voltage-controlled Ca²⁺ influx plays a major role in the regulation of the initial events of germination and filament growth. Increases in⁴⁵Ca²⁺ influx are observed after pretreatment of the aplanospores with low temperature shocks of brief duration or FCCP. Both agents are known to depolarize the surface membrane.⁴⁵Ca²⁺ influx is reduced in material treated with FC, an agent known to hyperpolarize cell membranes. The results indicate that Ca²⁺ influx takes place through voltage-sensitive Ca-channels.Abbreviations Bay K-8644 methyl 1,4-dihydro-2,6-dimethy1-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate - CGP CGP-28392 - CTC chlorotetracycline - dil diltiazem - DMSO dimethyl sulphoxide - DTE dithioerythritol - EGTA ethyleneglycol-bis-(-aminoethylether) N,N¹-tetraacetic acid - FC fusicoccin - FCCP p-fluoromethoxy-(carbonylcyanide)phenylhydrazone - nif nifedipine - PCMB p-chloromercuribenzoate - ver verapamil     

Contraction of human colonic circular smooth muscle cells is inhibited by the calcium channel blocker pinaverium bromide

The effects of L-type calcium channel blockers (CCBs) selective for the gastrointestinal tract (pinaverium) or non-selective (nicardipine and diltiazem), were investigated on CCK-, CCh- or KCl-induced contraction of smooth muscle cells (SMC) isolated from the circular muscle layer of normal or of inflamed human colons. In the normal tissue colon, whatever the contractile agent used, CCK-8 (1nM), CCh (1nM) or KCl (20mM), a micromolar concentration of pinaverium significantly inhibited contraction (88.36%, 93.10%, 93.92% inhibition respectively); this effect was concentration-dependent for CCh (IC50 = 0.73 +/- 0.08nM) and for CCK (IC50 = 0.92 +/- 0.12nM). In parallel, both nicardipine and diltiazem inhibit significantly contraction of isolated SMC. In inflamed colons, pinaverium (1 microM) display a significant higher efficacy than diltiazem or nicardipine to reduce cell contraction induced by CCK-8 or by KCl. In addition, RT-PCR experiments were performed to evidence tissue specificity of the L-type calcium channel. They revealed the expression of the messenger of the a-1 subunit L-type calcium channel (binding site of such CCBs), consistent with the expression of the rbC-2 splice variant of the alpha1-C gene.In conclusion: (i) the inhibition by calcium channel blockers of agonist-induced contractile activity suggest a modulation of SMC contraction upon extracellular calcium via 'L-type' voltage-dependent calcium channel; (ii) this study provides a rationale for the clinical use of pinaverium in colonic motor disoders affecting the contractility of SMC, since it appeared to decrease the contraction even in pathological situation; and (iii) RT-PCR experiments confirms the presence in human colon SMC of the alpha-1 subunit mRNA of calcium channel.     

The effects of calcium channel blockers on isoproterenol induced cyclic adenosine 3',5'-monophosphate generation in intact human lymphocytes

We have investigated the effects of clinically available calcium channel blockers (nifedipine, verapamil and diltiazem) on isoproterenol stimulated cyclic adenosine 3',5'-monophosphate (cyclic AMP) generation in intact human lymphocytes. After preincubation of various calcium antagonists with intact lymphocytes at 37 degrees C for 15 minutes, 10 microM nifedipine or verapamil partially inhibited isoproterenol induced cyclic AMP generation in the presence of cyclic AMP phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) while they alone had no effect on cyclic AMP level at a concentration of up to 100 microM. In contrast, 10 nM-1.0 microM nifedipine, verapamil or diltiazem potentiated cyclic AMP generation induced by isoproterenol in a dose dependent manner. Similar results were observed in the time course studies of cyclic AMP generation. These effects are somewhat similar to the effect of phenothiazine, a calmodulin inhibitor, which, at 10 microM (close to IC50), also potentiated the effects of isoproterenol. In contrast, lanthanum chloride (LaCl3), an extracellular inorganic calcium antagonist, at 1.0 mM, inhibited isoproterenol induced cyclic AMP generation. The biochemical mechanisms underlying these potentiating effects are unknown. It may be partly related to the effect of calcium channel blockers (at least for nifedipine) on preventing beta 2 adrenergic receptor desensitization. This may provide a potential mechanism for the synergistic effect between calcium channel blockers and beta 2 adrenoceptor agonists on bronchial dilatation.     

Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.     

Characterization of intestinal smooth muscle responses and binding sites for endothelin.

The contractile activity of and binding sites for endothelin-1 (ET-1) were investigated in isolated guinea-pig ileal longitudinal smooth muscle (GPILM). ET-1 produced concentration-dependent contractions of GPILM that either slowly subsided in the continued presence of ET-1 or rapidly subsided following washing of the tissue. The ED50 value for ET-1 contractions was 4.2 +/- 1.3 x 10(-9) M. The removal of extracellular calcium or pretreatment with nifedipine produced a complete inhibition of the contractions to ET-1. The IC50 value of nifedipine for inhibition of ET-1 mediated contractions was 3.0 +/- 0.8 x 10(-8) M. ET-1 produced a marked prolonged homologous desensitization of its contractile response but did not affect the responses mediated by carbachol, histamine, serotonin, substance P, and PLA2. High-affinity binding sites for 125I-labelled ET-1 were identified on microsomal membranes prepared from GPILM with Kd and Bmax values obtained by Scatchard analysis of 3.5 +/- 0.6 x 10(-10) M and 2138 +/- 159 fmol/mg protein, respectively. The binding of 125I-labelled ET-1 to GPILM microsomes was characterized by a rapid association (kob value of 0.077 min-1 at a radioligand concentration of 0.45 nM and an extremely slow dissociation (k1 value of 0.011 min-1; t1/2 value of 793 min). The binding was unaffected by the calcium channel antagonists nifedipine, verapamil, and diltiazem (10(-6) M); the receptor antagonists phenoxybenzamine, atropine, and naloxone (10(-6) M) and propranolol; and the peripheral benzodiazepine receptor antagonists Ro 5-4864 and PK 11195 and psychotomimetic drug phencyclidine (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

C(4)-alkyl substituted furanyl cyclobutenediones as potent, orally bioavailable CXCR2 and CXCR1 receptor antagonists

A novel series of cyclobutenedione centered C(4)-alkyl substituted furanyl analogs was developed as potent CXCR2 and CXCR1 antagonists. Compound 16 exhibits potent inhibitory activities against IL-8 binding to the receptors (CXCR2 Ki=1 nM, IC(50)=1.3 nM; CXCR1 Ki=3 nM, IC(50)=7.3 nM), and demonstrates potent inhibition against both Gro-alpha and IL-8 induced hPMN migration (chemotaxis: CXCR2 IC(50)=0.5 nM, CXCR1 IC(50)=37 nM). In addition, 16 has shown good oral pharmacokinetic profiles in rat, mouse, monkey, and dog.     

Dihydropyridine Binding Sites Regulate Calcium Influx Through Specific Voltage-Sensitive Calcium Channels in Cerebellar Granule Cells

In primary cultures of cerebellar granule cells, [3H]nitrendipine binds with high affinity to a single site (KD 1 nM and Bmax 20 fmol/mg protein). The 1,4-dihydropyridine (DHP) class of compounds such as nitrendipine, nifedipine, and BAY K 8644 displace [3H]nitrendipine binding at nanomolar concentrations. Verapamil partially inhibits whereas diltiazem slightly increases the [3H]nitrendipine binding. In these cells, the calcium influx that is induced by depolarization is very rapid and is blocked by micromolar concentrations of inorganic calcium blockers such as cadmium, cobalt, and manganese. The calcium influx resulting from cell depolarization is potentiated by BAY K 8644 and partially inhibited (approximately 40%) by nitrendipine and nifedipine. Other non-DHP voltage-sensitive calcium channel (VSCC) antagonists, such as verapamil and diltiazem, completely blocked the depolarization-induced calcium influx. This suggested that nitrendipine and nifedipine block only a certain population of VSCCs. In contrast, verapamil and diltiazem do not appear to be selective and block all of VSCCs. Perhaps some VSCCs can be allosterically modulated by the binding site for the DHPs, whereas verapamil and diltiazem may block completely the function of all VSCCs by occupying a site that differs from the DHP binding site.     

(+)-PN200-l 10 and Ouabain Binding Sites in Purified Bovine Adrenomedullary Plasma Membranes and Chromaffin Cells

Bovine adrenal medulla plasma membranes were purified by a differential centrifugation procedure using sucrose and Urografin discontinuous density gradients; the membranes were enriched 10-12-fold in acetylcholinesterase activity and [3H]ouabain binding sites. Specific (+)-[3H]PN200-110 binding to these membranes amounted to 90% of total binding and was saturable and of high affinity (KD = 41 pM; Bmax = 119 fmol/mg of protein) with a Hill coefficient close to 1, a result suggesting the presence of a single, homogeneous population of dihydropyridine receptors. The association and dissociation rate constants were, respectively, 7.5 X 108 M-1 min-1 and 0.023 min-1. Unlabeled (+)-PN200-110 displaced (+)-[3H]PN200-110 binding with a potency 100-fold higher than (-)-PN200-110 (IC50,0.5 and 45nM, respectively). Although the two enantiomers of BAY K 8644 completely displaced (+)-[3H]PN200-110 binding, they exhibited no stereoselectivity (IC50, 69 and 83 nM,respectively). Whereas ( +/- )-nitrendipine very potently displaced (+)-[3H]PN200-110 binding (IC50 = 1.3 nM) verapamil and cinnarizine displaced the binding by only 30 and 40% at 1 microM, and diltiazem increased it by 20% at 10 microM. [3H]Ouabain bound to plasma membranes with a KD of 34 nM and a Bmax of 9.75 pmol/mg of protein, a figure 80-fold higher than the Bmax for (+)-PN200-110. [3H]Ouabain also bound to intact chromaffin cells with a Bmax of 244 fmol/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Channels involved in transient currents unmasked by removal of extracellular calcium in cardiac cells

In cardiac cells that lack macroscopic transient outward K(+) currents (I(to)), the removal of extracellular Ca(2+) can unmask "I(to)-like" currents. With the use of pig ventricular myocytes and the whole cell patch-clamp technique, we examined the possibility that cation efflux via L-type Ca(2+) channels underlies these currents. Removal of extracellular Ca(2+) and extracellular Mg(2+) induced time-independent currents at all potentials and time-dependent currents at potentials greater than -50 mV. Either K(+) or Cs(+) could carry the time-dependent currents, with reversal potential of +8 mV with internal K(+) and +34 mV with Cs(+). Activation and inactivation were voltage dependent [Boltzmann distributions with potential of half-maximal value (V(1/2)) = -24 mV and slope = -9 mV for activation; V(1/2) = -58 mV and slope = 13 mV for inactivation]. The time-dependent currents were resistant to 4-aminopyridine and to DIDS but blocked by nifedipine at high concentrations (IC(50) = 2 microM) as well as by verapamil and diltiazem. They could be increased by BAY K-8644 or by isoproterenol. We conclude that the I(to)-like currents are due to monovalent cation flow through L-type Ca(2+) channels, which in pig myocytes show low sensitivity to nifedipine.     

Solubilization of Calcium Channel Antagonist Binding Sites from Rat Brain

Calcium antagonist binding sites were solubilized from rat brain membranes using the detergent 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate (CHAPS). CHAPS-solubilized [3H]nitrendipine binding sites are saturable over a range of 0.05-4 nM and Scatchard analysis reveals a single, high-affinity (KD = 0.49 +/- 0.10 nM), low-capacity (Bmax = 56 +/- 4 fmol/mg of protein) binding site. Reversible ligand competition experiments using solubilized binding sites demonstrated appropriate pharmacologic specificity, with dihydropyridines (nifedipine = nitrendipine greater than Bay K 8644) completely displacing binding, verapamil partially displacing binding, and diltiazem enhancing binding, as previously described in membrane preparations. Lyophilized Crotalus atrox venom was purified by ion exchange chromatography followed by gel filtration to a single peptide band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This fraction of molecular weight 60,000 competitively inhibits [3H]nitrendipine binding to both membrane and soluble preparations with an IC50 of 5 micrograms/ml. This polypeptide should serve as a useful ligand for future efforts in purifying the dihydropyridine calcium channel binding site in brain.     

IL-1alpha but not IL-1beta-induced prostaglandin synthesis is inhibited by corticotropin-releasing factor

Interleukin (IL)-1alpha and IL-1beta share low amino acid homology, but exhibit a very similar array of biological activities. The authors previously showed negative regulation of IL-1alpha-induced prostaglandin (PG) production by corticotropin releasing factor (CRF). In this study, the authors compared the effect of CRF on IL-1alpha- and IL-1beta-induced PG synthesis. IL-1alpha (100 U/ml) increased prostacyclin (PGI2) (measured as 6-keto PGF1alpha[6K]) synthesis in endothelial cells and the production of PGE2in fibroblasts. The PG response to IL-1alpha was suppressed by simultaneous exposure to CRF (2.5x10(-11)-2.5x10(-8) M) in both cell types. IL-1alpha enhanced both phospholipase A2(PLA2) and prostaglandin H synthase (PGHS) activities, and the two effects were completely abrogated by CRF. IL- 1beta (100 U/ml) was as active as IL-1alpha in triggering release of PGI2 from endothelial cells and PGE2 from fibroblasts. However, CRF (2.5x10(-11)-2.5x10(-8) M) failed to alter the IL-1beta-induced PG synthesis in both cell types. Following IL-1beta PGHS activity, and to a lesser extent PLA2 activity, were enhanced, however CRF only inhibited PGHS and not PLA2 activity. It is concluded that although IL-1alpha and IL-1beta usually produce similar biological effects, here they seem to act via different mechanisms. The different regulation of IL-1alpha and IL-1beta pro-inflammatory activities by CRF may attribute special precision and specificity to the neuroendocrine-immune control of inflammatory processes.     

Attenuation of daunorubicin-augmented microsomal lipid peroxidation and oxygen consumption by calcium channel antagonists.

Daunorubicin (20 microM) stimulated NADPH-dependent microsomal lipid peroxidation about 2-fold over control values and enhanced the rate of oxygen utilization by microsomes. The calcium channel blockers tested inhibited daunorubicin-augmented lipid peroxidation and O2 consumption to varying degrees. Inhibition of daunorubicin-stimulated lipid peroxidation was found to be dose dependent; the IC50 (drug concentration producing 50% inhibition of lipid peroxidation) values for verapamil, nifedipine and diltiazem were approximately 150 microM, 200 microM, and 600 microM respectively. Our in vitro studies suggest that calcium channel antagonists may modulate the free radical-mediated, cardiotoxic effects of daunorubicin.     

Activated Rho/Rho kinase and modified calcium sensitivity in cryopreserved human saphenous veins

Background

We have shown previously that cryopreservation of human internal mammary arteries activates protein kinase C and enhances intracellular Ca²⁺ [Ca²⁺]_i. We now present evidence that in human saphenous veins (HSV) cryoinjury is associated with activation of the Rho/Rho kinase signaling pathways and enhanced [Ca²⁺]_i.

Methods

HSV were investigated in vitro either unfrozen within 12 h after removal or after storage at −196 °C in a cryomedium containing 1.8 M dimethyl sulfoxide and 0.1 M sucrose as cryoprotectant additives.

Results

Cryostorage diminished responses to receptor-mediated contractile agonists such as noradrenaline, 5-HT and endothelin-1 by up to 30% whereas responses to KCl were attenuated by about 50%. Concentration-response curves for CaCl₂ on unfrozen and cryopreserved HSV revealed similar inhibitory activities of both blocking 1,4-dihydropyridine derivatives nifedipine and the (−)-(R) enantiomer of SDZ 202-791 whereas the Ca²⁺ channel activating (+)-(S) enantiomer of SDZ 202-791 was 10 times less effective at enhancing contractions to CaCl₂ when tested after cryostorage. These functional effects were reflected by changes in [Ca²⁺]_i as demonstrated by fluorescence of Fluo-3AM loaded veins. The diminished activity of (+)-(S) SDZ 202-791 in cryopreserved HSV was reversed partially when the potassium channel opener pinacidil (1 μM) was present during the freezing/thawing process. Blockade of Rho kinase by HA-1077 proved to be significantly more effective at attenuating contractile responses to both endothelin-1 and KCl after cryostorage.

Conclusions

Data suggested that cryopreservation modified [Ca²⁺]_i of venous smooth muscle cells (1) through depolarization-induced changes in Ca²⁺ influx and (2) through activation of Rho kinase signaling pathways.     

Activation of purified cardiac ryanodine receptors by dihydropyridine agonists

Prior observations have raised the possibility that dihydropyridine (DHP) agonists directly affect the sarcoplasmic reticulum (SR) cardiac Ca(2+) release channel [i.e., ryanodine receptor (RyR)]. In single-channel recordings of purified canine cardiac RyR, both DHP agonists (-)-BAY K 8644 and (+)-SDZ202-791 increased the open probability of the RyR when added to the cytoplasmic face of the channel. Importantly, the DHP antagonists nifedipine and (-)-SDZ202-791 had no competitive blocking effects either alone or after channel activation with agonist. Thus there is a stereospecific effect of SDZ202-791, such that the agonist activates the channel, whereas the antagonist has little effect on channel activity. Further experiments showed that DHP agonists changed RyR activation by suppressing Ca(2+)-induced inactivation of the channel. We concluded that DHP agonists can also influence RyR single-channel activity directly at a unique allosteric site located on the cytoplasmic face of the channel. Similar results were obtained in human purified cardiac RyR. An implication of these data is that RyR activation by DHP agonists is likely to cause a loss of Ca(2+) from the SR and to contribute to the negative inotropic effects of these agents reported by other investigators. Our results support this notion that the negative inotropic effects of DHP agonists result in part from direct alteration in the activity of RyRs.