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  • 1
    ISSN: 1432-2013
    Keywords: Scorpion toxin ; Myelinated nerve fiber ; pH ; Voltage clamp
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract 1. New toxins, VI and VII, were purified from the venom of the North American scorption,Centruroides sculpturatus, and studied in the voltage-clamped frog node of Ranvier. These toxins reduced peak inward Na currents and caused a transient, depolarization-induced shift in the voltage dependence of Na activation. Their effects were indistinguishable from those of toxins I, III and IV, as previously described by Meves et al. (1982). 2. Toxin VII, 0.2 μg/ml, shifted the voltage dependence of steady-state inactivation (h ∞). In four fibers, the mean shift of theh ∞(E) curve was −17 mV, compared to a mean shift of −28 mV in the descending branch of theI Na(E) curve. Theh ∞(E) curve with toxin VII was monotonic and inactivation was incomplete at positive potentials. 3. Decreasing the pH from 7.4 to 5.7 increased the shift in the voltage dependence of activation with toxin. The increase with toxin VI at pH 5.7 was reversible on returning to pH 7.4, but the increase with toxin VII was not. The lack of reversibility of the effect of pH with toxin VII was quantified in two ways: (a) the mean value ofI Na (measured at −62 mV with a conditioning pulse) at pH 7.4 was 5.1 times larger after treatment than before treatment with 0.1 μg/ml of toxin VII at pH 5.7; (b) the average concentration of toxin VII required for a shift of −30 mV at pH 7.4 was decreased by a factor of 4.3, to 0.19 μg/ml (26 nM), by pretreatment at pH 5.7. In comparison, a shift of −30 mV was produced with 2.31 μg/ml (319 nM) of toxin VI. 4. For both toxin VI and toxin VII (in the latter case, after initial pretreatment with toxin at pH 5.7), the shift in the voltage dependence of activation with a conditioning pulse was 1.8 times larger at pH 5.7 than at pH 7.4. The increase was accounted for by an essentially normal shift (mean = +13.1 mV;n=7) of theI Na(E) curve without a conditioning pulse at pH 5.7, but a minimal shift (mean = +0.4 mV) of theI Na(E) curve with a conditioning pulse. Permeability ratios at the two pH's reached the value of 0.5 at a membrane potential of +28 mV without a conditioning pulse, whereas this value was reached near −62 mV when a conditioning pulse preceded the test pulse. 5. These observations were interpreted as evidence that the binding of both toxin VI and toxin VII is greater at pH 5.7 than at pH 7.4, but that the increase with toxin VI is reversible on increasing the pH whereas the increase with toxin VII is not under the experimental conditions used.
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  • 2
    ISSN: 1432-2013
    Keywords: Key words Ca2+ channel ; Smooth muscle cell ; Basilar artery ; Nitric oxide ; Sodium nitroprusside ; cGMP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  We studied the effect of the nitric oxide (NO) donor, sodium nitroprusside (SNP), on the macroscopic and single-channel currents due to the 22-pS Ca2+ channel in smooth muscle cells from guinea pig basilar artery. In nystatin-perforated whole-cell recordings, 50 nM SNP decreased the macroscopic current to 63±12% of control values, without changing the voltage dependence of the current. In cell-attached patches with BAY-K8644 in the pipette, SNP caused a comparable decrease in single-channel availability (n ·P o) that was dose dependent over the range of 10 nM to 10 μM SNP. SNP had no effect on single-channel properties, including slope conductance, voltage dependence of activation, the number of open states, the time constants of the open states, and the proportion of time spent in each open state. The effect of SNP (50 nM) on single Ca2+ channel openings was reproduced by 8-Br-cGMP (100 μM), which also reduced channel availability without altering channel properties. The protein kinase inhibitor H-8 (1.5 μM), which exhibits relative specificity for cGMP-dependent protein kinase, completely inhibited the decrease in single-channel availability expected with SNP. The dose-dependent decrease in Ca2+ channel availability caused by SNP was not altered by prior application of 8-Br-cAMP or forskolin, both of which cause an increase in Ca2+ channel availability in these cells. Our findings suggest that NO decreases openings of Ca2+ channels in basilar artery smooth muscle cells without altering channel properties, and that it does so by a mechanism likely to involve cGMP-dependent protein kinase.
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  • 3
    ISSN: 1432-2013
    Keywords: Ca2+-activated K+ channel ; BK channel ; Isoproterenol ; β-Adrenoceptor ; Protein kinase A ; Smooth muscle cell ; Basilar artery ; Smooth muscle relaxation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We studied the effect of isoproterenol on the Ca2+-activated K+(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca2+ concentration, [Ca2+]i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from aV H of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca2+]i from 0.01 to 1.0 μM. With [Ca2+]i between 0.1 and 1.0 μM, 0.4 μM isoproterenol increased this current by 58.6±17.1%, whereas with [Ca2+]i at 0.01 μM a sixfold smaller increase was observed. With [Ca2+]i≥0.1 μM, 100 μM dibutyryl-adenosine 3′:5: cyclic monophosphate (cAMP) and 1 μM forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 μM propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from aV H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca2+]i from 0.01 to 1.0 μM. Outside-out-whole-cell recordings with [Ca2+]i≥0.1 μM indicated that 100 μM dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca2+]i, with a 35-fold larger effect observed with 0.1–0.5 μM Ca2+ compared to 0.01 μM Ca2+. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated byβ-adrenoceptor stimulation, that the effect depends strongly on [Ca2+]i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate inβ-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.
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  • 4
    ISSN: 1432-2013
    Keywords: Ca2+ channel ; Smooth muscle cell ; Basilar artery
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We studied single Ca2+ channels in smooth muscle cells from the basilar artery of the guinea pig using conventional patch-clamp techniques. With 40 mM or 90 mM Ba2+ as the charge carrier, a 23-pS inward current channel was observed in 46/187 cell-attached patches studied without the dihydropyridine, BAY K8644, in the pipette solution. At 0 mV, this channel exhibited short and long openings with time constants of 1.03 and 3.65 ms, respectively. The probability of channel opening was voltage dependent with half-activation occurring at +9.9 mV. In 14/26 patches tested, addition of 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) to the bath increased the probability of opening at -10 mV by a factor of 2.6, from 0.0272±0.0429 to 0.0695±0.0788 (P 〈0.01, paired t-test). Mean data from five patches fit to a Boltzmann function indicated that at positive potentials, the probability of opening increased by a factor of 1.7, from 0.352 to 0.600, whereas the voltage dependence, the number of channels, the number of open states, the time constants of the open states, and the proportion of time spent in each open state were unchanged. When BAY K8644 was added to the pipette solution, the 23-pS channel was observed in nearly all patches (62/66), but the voltage dependence of activation was shifted −15.3 mV compared to control. In some patches studied with 90 mM Ba2+, a 9-pS inward current channel also was observed and its activity also was increased significantly by 8-Br-cAMP. When membrane patches were excised from the cell and studied in an inside-out configuration, single-channel activity due to the 23-pS channel lasted 1–3 min before being irreversibly lost, regardless of the presence of BAY K8644 in the pipette or of 8-Br-cAMP plus Mg · ATP and leupeptin in the bath. Subsequent addition of the catalytic subunit of protein kinase A (PKACS) did not restore Ca2+ channel activity. Conversely, when patches were excised into a solution already containing 8-Br-cAMP plus Mg · ATP, leupeptin and PKACS, channel activity was prominent and generally lasted until the seal was lost, or until the experiment was terminated at 30–45 min, unless protein kinase inhibitor also was present, in which case channel lifetime was short. Our findings indicate that availability of the L-type Ca2+ channel in basilar artery smooth muscle cells is increased by activation of cAMP-dependent protein kinase A, and that the (or one of the) phosphoprotein(s) involved may not be membrane bound.
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  • 5
    ISSN: 1432-2013
    Keywords: calcium channels ; smooth muscle cells ; cerebral arterioles ; basilar artery
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We compared Ca2+ channels in cell-attached patches of smooth muscle cells from cerebral precapillary arterioles and basilar artery of guinea pig. Patches were studied without Ca2+ channel activators in the pipette solution. In both preparations, a 23 pS channel (40 mM Ba2+) sensitive to block by nifedipine was identified. In arteriolar but not in basilar artery patches, channel activity was recorded without apparent inactivation at potentials of −40 to −20 mV. Values for the number of channels in a patch x probability of channel opening (n·Po) at various potentials were fit to a Boltzmann function. For the arteriolar patches (n=5) and for patches from basilar artery (n=5), the midpoint potentials for the voltage dependence of n·Po were −9.3 mV and +8.9 mV, and maximum values of n·Po at positive potentials were 1.23 and 0.33. At potentials ≥0 mV, the average for the maximum number of superimposed openings in basilar artery patches was 1.7 (n=17) and in arteriolar patches was 6.5 (n=6). For both preparations, histograms of channel open times at −10 mV required two time constants, 0.48 and 3.95 ms, and the shorter open state accounted for 88% of openings. Our data indicate that Ca2+ channel activity is likely to be more prominent near resting membrane potentials in arteriolar cells than in basilar artery cells.
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  • 6
    ISSN: 1432-2013
    Keywords: Calcium channel ; Smooth muscle cell ; Basilar artery
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Ca2+ channel currents were studied in smooth muscle cells from the basilar artery of the guinea pig using the whole-cell patch-clamp technique, 10 mM Ba2+ as the charge carrier and strong buffering of intracellular Ca2+ with EGTA (pCai=8). Cell capacitance was 18.8 ± 6.6 pF (n= 96) and maximum current density at +10 to +20 mV (holding potential 〈 −55 mV), measured early in dialysis, was −14.8 ± 4.9 pA/pF (n= 83). Currents reversed at approximately +95 mV and, at more positive potentials, outward Cs+ currents were recorded that were blocked by either external Cd2+ or Ca2+. One component of current was identified that had properties consistent with L-type channels. On the basis of measurements of tail currents, its threshold for activation was −15 mV, its voltage dependence of activation was steep and it was half-activated at +8.5 mV. It inactivated very slowly at +15 mV (2787 ± 511 ms) and it deactivated rapidly (251 ± 55 μs) at −55 mV. It was quickly lost during dialysis and was largely blocked by 1 nM nifedipine (1-s pulses, holding potential = −55 mV). A second component, termed B-type current, was identified that had properties inconsistent with those of T-type channels. On the basis of tail currents, its threshold for activation was −30 mV, its voltage dependence of activation was less steep and it was half-activated at +33.7 mV. It was half-inactivated at −32.1 mV, it inactivated with a time constant of 162 ± 13 ms at +15 mV and it deactivated relatively slowly (3.8 ± 0.8 ms) at −55 mV. It was less sensitive than L-type current to dialysis and to block by nifedipine.
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  • 7
    ISSN: 1432-0878
    Keywords: Muscle, smooth ; Cerebral blood vessels ; Cell culture CNS ; Bulk isolation ; Guinea pig (Rodentia)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Published methods for the isolation of cerebral microvessels primarily yield terminal resistance vessels and capillary networks, not the more proximal, subpial penetrating arterioles desired for certain studies. We report a novel method for isolating microvessels from the cerebral cortex of a single guinea-pig brain that yields large arteriolar complexes that are up to 50% intact. Instead of using homogenization to disperse brain parenchyma, we digested cortical fragments with trypsin, gently dispersed the parenchyma mechanically, and recovered microvascular complexes by sieving. Phase-contrast and electron microscopy showed primary (penetrating) arterioles, secondary arterioles, and capillary networks that frequently were in continuity as intact microvascular units. Culture of microvascular cells was carried out by enzymatic dissociation followed by an overnight incubation in a recovery medium at 4°C before plating onto fibronectin-modified surfaces. Viability of isolated cells was demonstrated by good cell attachment and prompt proliferation that resulted in confluent cultures after 10 days. Confluent secondary cultures demonstrated characteristic features of smooth muscle cells, including a “hill-and-valley” growth pattern and expression of α-actin. Less than 1% of cells were endothelial or astrocytic cells by immunocytochemical and morphologic criteria. Ultrastructural studies demonstrated evidence of a synthetic phenotype of smooth muscle cell and absence of a significant number of fibroblasts. This method demonstrates that viable smooth muscle cells from the cerebral parenchymal microvasculature can be isolated in bulk quantities for study in vitro.
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  • 8
    ISSN: 1432-1920
    Keywords: Key words Interventional neuroradiology ; Papaverine ; Subarachnoid haemorrhage ; Vasospasm
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We assessed the prevalence of recurrent vasospasm following failure of intra-arterial papaverine and the efficacy of repeat intra-arterial infusions of papaverine for control of recurrent vasospasm. Of 24 patients treated with intra-arterial papaverine for vasospasm following aneurysm surgery, 12 did not improve clinically after the initial treatment; 9 received second or third infusions on consecutive days; 6 received only a second infusion; and 3 received a third. Superselective infusion into the intracranial arteries was performed in all nine cases. Despite angiographic improvement after the initial or second infusions, all nine patients showed varying degrees of recurrent vasospasm at the time of the second or third treatment. Within 24 h of a second infusion, three of the six patients had significant clinical improvement, and one of these showed marked improvement soon after a third infusion. Our preliminary results suggest that repeat papaverine infusion may be a way of controlling recurrent or recalcitrant vasospasm.
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