Dispersed Single Smooth Muscle Cells Research Articles

Pharmacological and molecular evidence for the involvement of Kv4.3 in ultra‐fast activating K+ currents in murine portal vein myocytes

The aim of this study was to determine the molecular identity of a transient K+ current (termed IUF) in mouse portal vein myocytes using pharmacological and molecular tools. Whole cell currents were recorded using the ruptured patch con from either acutely dispersed single smooth muscle cells from the murine portal vein or human embryonic kidney cells. Reverse transcriptase polymerase reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse portal vein using primers specific for various voltage-dependent K+ channels, auxillary subunits and calcium-binding proteins. Immunocytochemistry was undertaken using an antibody specific for Kv4.3. IUF had a mean amplitude at +40 mV of 558 +/- 50 pA (n = 32) with a mean time to peak at +40 mV of approximately 4 ms. IUF activated and inactivated with a half maximal voltage of -12 +/- 2 mV and -85 +/- 2 mV, respectively. IUF was relatively resistant to 4-aminopyridine (5 mM produced 30 +/- 6 % block at +20 mV) but was inhibited effectively by flecainide (IC50 value was 100 nM) and phrixotoxin II. This pharmacological profile is consistent with IUF being comprised of Kv4.x proteins and this is supported by the results from the quantitative PCR and immunocytochemical experiments. These data represent a rigorous investigation of the molecular basis of vascular transient K+ currents and implicates Kv4.3 as a major component of the channel complex.

ANG II and vasopressin stimulate calcium entry in dispersed smooth muscle cells of preglomerular arterioles.

Calcium signaling mechanisms were examined in vessel segments and dispersed single smooth muscle cells (SMC) of interlobular arteries and afferent arterioles (< 50 microns diameter) from the rat kidney. These resistance vessels were isolated from rat kidneys, using an iron oxide-sieving technique with subsequent collagenase digestion. Individual cells were identified by their characteristic oval appearance and positive staining for smooth muscle-specific alpha-actin and heavy chain myosin SM-1 and SM-2. Cytosolic calcium concentration ([Ca2+]i) was measured using fura 2 ratiometric fluorescence at 340 and 380 nm wavelength with a microscope-based photometer. Angiotensin II (ANG II) and arginine vasopressin (AVP), at concentrations of 10(-10)-10(-6) M, produced dose-dependent increases in [Ca2+]i; maximum increases were 221 +/- 49 nM for ANG II and 237 +/- 49 nM for AVP. The temporal response patterns for both agonists were characterized by a square-shaped, immediate step increase in [Ca2+]i to a near maximum level that was maintained through the recording period of 150-200 s. Responses of individual dispersed SMC and short vessel segments were similar. Losartan antagonized the action of ANG II, indicating mediation by AT1 receptors on preglomerular arteriolar SMC. The V1-selective antagonist [d(CH2)5Tyr(Me)2Tyr(NH2)9]AVP completely inhibited AVP-induced [Ca2+]i changes. The importance of calcium entry in hormone-induced changes in [Ca2+]i was demonstrated by the finding that neither ANG II nor AVP elicited a [Ca2+]i response in media rendered nominally calcium free by addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Calcium entry occurred primarily through L-type, voltage-gated calcium channels as the dihydropyridine, nifedipine, completely prevented or reversed [Ca2+]i changes normally elicited by either hormone. Our results provide new information about the similarity of calcium signaling in single SMC and short segments freshly isolated from renal interlobular arteries and afferent arterioles. The observations indicate that AT1 and V1 receptors are coupled to signal transduction pathways leading to rapid changes in [Ca2+]i. Calcium mobilization appears to play a minor to nonexistent role under the experimental conditions. The predominant mechanism involves calcium entry through dihydropyridine-sensitive, voltage-gated calcium channels in single SMC from these resistance vessels.

Ca2+ release mechanism studied in single isolated smooth muscle cells

This paper briefly reviews our current effort to study the Ca2+ mobilization mechanism in enzymatically dispersed single smooth muscle cells. Each single cell obtained from guinea pig taenia caeci possesses two types of Ca2+ stores, one (S alpha) with both Ca(2+)-induced and IP3-induced Ca2+ release mechanisms and the other (S beta) with only IP3-induced Ca2+ release mechanism. After depletion of S alpha either with ryanodine treatment or with caffeine pretreatment, carbachol failed to induce Ca2+ release, while intracellular application of IP3 did induce Ca2+ release. Our results suggest that the difference between the agonist- and IP3-induced responses can be resolved by obligatory involvement of positive feedback control of IP3-induced Ca2+ release in the agonist-induced Ca2+ release. Furthermore, we were able to demonstrate that the dose-response relation in single cells shows an all-or-none feature, which seems at least partly due to the feedback control of Ca2+ release. We discuss the reasons why graded dose-response relation is obtained in bundles of smooth muscles, while the response of single cells is an all-or-none type.

Effects of ketamine on voltage-dependent Ca2+ currents in single smooth muscle cells from rabbit portal vein.

The effects of ketamine on membrane potentials and long-lasting type (L-type) voltage-dependent Ca2+ currents were investigated in dispersed single smooth muscle cells from rabbit portal veins. The amplitude and duration of the action potentials evoked by intracellular stimulation were inhibited by ketamine and were completely blocked by 10(-3) mol/l ketamine. Ketamine, however, did not alter the resting membrane potential. Whole cell voltage clamp experiments revealed that ketamine at concentrations higher than 10(-4) mol/l reduced the peak L-type voltage-dependent Ca2+ currents. These effects of ketamine were concentration-dependent and reversible at concentrations from 10(-4) to 10(-3) mol/l. Moreover, the activation threshold of L-type Ca2+ current (approximately -30 mV) was slightly shifted to the positive potential side by ketamine. This effect can explain that the action potential was abolished by 10(-3) mol/l ketamine. It is concluded that relaxation of the vascular smooth muscle by ketamine may have to be attributed to the inhibition of L-type voltage-dependent Ca2+ current.

Inward current in single smooth muscle cells of the guinea pig taenia coli.

Using the tight-seal voltage-clamp method, the ionic currents in the enzymatically dispersed single smooth muscle cells of the guinea pig taenia coli have been studied. In a physiological medium containing 3 mM Ca2+, the cells are gently tapering spindles, averaging 201 (length) x 8 microns (largest diameter in center of cell), with a volume of 5 pl. The average cell capacitance is 50 pF, and the specific membrane capacitance 1.15 microF/cm2. The input impedance of the resting cell is 1-2 G omega. Spatially uniform voltage-control prevails after the first 400 microseconds. There is much overlap of the inward and outward currents, but the inward current can be isolated by applying Cs+ internally to block all potassium currents. The inward current is carried by Ca2+. Activation begins at approximately -30 mV, maximum ICa occurs at +10-+20 mV, and the reversal potential is approximately +75 mV. The Ca2+ channel is permeable to Sr2+ and Ba2+, and to Cs+ moving outwards, but not to Na+ moving inwards. Activation and deactivation are very rapid at approximately 33 degrees C, with time-constants of less than 1 ms. Inactivation has a complex time course, resolvable into three exponential components, with average time constants (at 0 mV) of 7, 45, and 400 ms, which are affected differently by voltage. Steady-state inactivation is half-maximal at -30 mV for all components combined, but -36 mV for the fast component and -26 and -23 mV for the other two components. The presence of multiple forms of Ca2+ channel is inferred from the inactivation characteristics, not from activation properties. Recovery of the fast channel occurs with a time-constant of 72 ms (at +10 mV). Ca2+ influx during an action potential can transfer approximately 9 pC of charge, which could elevate intracellular Ca2+ concentration adequately for various physiological functions.

Regulation of calcium current by intracellular calcium in smooth muscle cells of rabbit portal vein.

Effects of concentrations of intracellular calcium, [Ca2+]i, on the voltage-dependent Ca2+ current (ICa) recorded from dispersed single smooth muscle cells of the rabbit portal vein were studied, using a whole cell voltage clamp method combined with an intracellular perfusion technique. Outward currents were minimized by replacement of Cs+ -rich solution in the pipette and 20 mM tetraethylammonium in the bath. The ICa was evoked by command pulses of above -30 mV, and the maximum amplitude was obtained at about 0 mV. This ICa was dose dependently inhibited by increases in the [Ca2+]i above 30 nM. The Kd value of the [Ca2+]i required to inhibit the ICa was about 100 nM. The Ba2+ current was also inhibited by increases in the [Ca2+]i. Conversely, perfusion of Ba2+ into the cell up to 100 microM did not suppress the ICa. Changes in the [Ca2+]i did not modify the steady-state inactivation curve. The inhibition of the ICa evoked by the test pulse is most prominent when the preceding influx of Ca2+ during the conditioning pulse was large, as estimated using a double pulse protocol. This inhibition was proportionally reduced by increases in the concentration of the Ca2+ chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Therefore, the Ca2+ -dependent inactivation of the Ca2+ channel may contribute toward regulating [Ca2+]i in smooth muscle cells of the rabbit portal vein.

Acetylcholine activates single sodium channels in smooth muscle cells

Using the whole-cell clamp technique with a patch electrode, single channel activities were recorded in dispersed single smooth muscle cells of the guinea-pig ileum in response to application of greater than 1 microM acetylcholine (ACh) or carbachol (CCh). Under physiological conditions (bath:Krebs, pipette:High K), these channels have a single channel conductance of 20-25 pS at the membrane potentials ranging between -100 and -40 mV and are activated in the membrane potential dependent manner. The amplitude of the channel current showed a strong dependence on the extracellular Na concentration but the reversal potential obtained by the extrapolation of the I-V relationship was not consistent with the equilibrium potential of Na ion. An approximate estimation of permeability ratio based on the independent principle described by Hodgkin et al. gave a value of Na:K = 1.0:0.3-0.4 to this channel. From features of the macroscopic and single channel currents, it is concluded that these muscarinic ACh(CCh)-activated channels mainly pass Na ion and play a major part in the membrane depolarization produced by ACh or CCh in mammalian intestinal smooth muscles.

Features of 4-aminopyridine sensitive outward current observed in single smooth muscle cells from the rabbit pulmonary artery.

The 4-aminopyridine (4AP) sensitive outward current of enzymatically dispersed single smooth muscle cells of the rabbit main pulmonary artery were investigated using the voltage clamp method. When the cell was exposed to physiological salt solution (PSS) in the bath and high K+ in the pipette no inward current was generated by depolarization of the membrane, but when 4AP was present in the bath or when Cs+ with tetraethylammonium+ (Cs+-TEA+) in the pipette, an inward current was generated. This current was enhanced by Ba2+ or high Ca2+ and was blocked by inorganic or organic Ca2+ channel blockers. The outward current was partly inhibited by the Ca2+ channel blockers, Ca2+-free or Mn2+ containing solution. The residual outward current was blocked by external application of 10 mM 4AP, whereas it was inhibited by half with 100 mM TEA+. To investigate further natures of 4AP sensitive outward current, the following experiments were done in the bath solution containing 2.5 mM Mn2+. The reversal potential of this outward current, estimated from the tail current, remained the same in Na+-deficient solution, but shifted to near the K+-equilibrium potential in Cl- deficient solution. Thus, the main current carrier for the outward current seems to be K+, but Cl- may participate to some extent. The amplitude of the outward current decreased slowly. However, the reversal potential was not changed, suggesting the reduction in amplitude of the outward current was not due to the accumulation of K+ on the outer surface of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of adrenergic agonists on Ca2+-channel currents in single vascular smooth muscle cells

Ca2+-channel currents have been measured in enzymatically dispersed single smooth muscle cells of the rabbit ear artery using the whole-cell patch clamp technique. Inward currents were elicited by depolarizing test pulses from a holding potential of -50 mV. These currents were activated from -30 mV onward and reached full activation around 0 mV. alpha-Adrenergic agonists did not affect the background current measured at the holding potential, but markedly reduced the peak amplitude of the voltage-activated Ca2+-channel currents. This alpha-adrenergic inhibition also occurred in cells which were internally perfused with solutions containing either 10 microM cAMP, 10 microM cGMP or 0.1 mM GTP, but became irreversible when the pipette solution contained a non-hydrolyzable GTP-analog. The action of beta-agonists on the voltage-activated Ca2+-channel currents was variable, and ranged from no effect at all to a 50% reduction of the current. It is concluded that alpha-agonists do not open receptor-operated Ca2+-channels in these smooth muscle cells. The inhibition of the voltage-activated Ca2+-currents does not seem to be mediated through changes in cyclic nucleotide levels, but might be mediated through G-proteins. Its physiological relevance remains however unclear. The action of beta-agonists is consistent with their relaxing effect, but the reason for the non-uniform response has not been elucidated.

Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of the rabbit.

1. Whole-cell membrane current recordings under voltage clamp were made at room temperature from dispersed single cells of longitudinal smooth muscle of rabbit jejunum and dispersed single smooth muscle cells of rabbit ear artery using patch pipettes containing up to 10 mM-EGTA Ca buffer. 2. Spontaneous transient outward currents (s.t.o.c.s) up to 250 pA in size and about 100 ms in duration were observed in conditions which might lead to an elevated internal Ca concentration. The amplitude distribution in some cells and form of the currents suggested that they were evoked by a quantal stimulus. 3. S.t.o.c. amplitude was voltage dependent and reversed at the K equilibrium potential. S.t.o.c.s. were blocked by 1 mM-tetraethylammonium or 10 mM-Ba applied externally or by perfusing Cs inside the cell. 4. Removing external Ca abolished s.t.o.c. activity in the jejunal cells but not in arterial cells. Increasing EGTA buffering within the cells from 1 mM or less to 10 mM abolished activity in both cell types. 5. Caffeine (5 mM) applied to the bathing solution stimulated rapid discharge of transient outward currents and then a prolonged period of inhibition. The stimulated discharge was sensitive to external Ca in jejunal cells but much less so in arterial cells. ACh applied by ionophoresis to jejunal cells or noradrenaline bath-applied onto arterial cells also stimulated discharge of transients followed by a prolonged inhibitory phase. 6. It was suggested that s.t.o.c.s represent the simultaneous opening of up to 75-100 Ca-activated K channels at -40 mV in response to sudden discharge of Ca from internal stores when these became overloaded, and that this process may occur cyclically.

Calcium‐activated potassium channels in single smooth muscle cells of rabbit jejunum and guinea‐pig mesenteric artery.

Single-channel studies were made using the patch-clamp technique of K channels in dispersed single smooth muscle cells from rabbit longitudinal jejunal muscle and guinea-pig small (less than 0.2 mm o.d.) mesenteric arteries. In isolated inside-out patches from these two types of smooth muscle cell there was a population of K channels which had single-channel conductances of about 100 pS in near physiological K gradients and about 200 pS with symmetrical 126 mM-K solutions. Their conductance and other properties distinguish them from a K channel of smaller conductance which we have previously described in these cells. The relative permeability of the channel with respect to K was 1.4 Tl:1.0 K:0.7 Rb: less than 0.05 Na: less than 0.05 Cs. Cs (1 mM applied to the outside of the membrane) interfered with inward K movement when the membrane was hyperpolarized. Rb conductance of the channel when both sides of the membrane were exposed to 126 mM-Rb was 30 pS. When the Ca concentration on the inside of the membrane ([Ca]i) was about 10(-9) M, K channel opening was rarely observed and then only at strongly positive potentials. At [Ca]i between 10(-9) M and 10(-7) M mean channel open time increased and the probability of channel opening increased steeply; both were further increased by increasing membrane positivity. At [Ca]i between 10(-6) M and 2.5 mM the channel was mainly in the open state and the probability of channel conducting state often declined with increasing membrane positivity. The effects of varying [Ca]i from 10(-7) M to 2.5 mM on the kinetic activity of a single channel was studied largely in mesenteric artery patches containing one active channel. The distribution of open times could be fitted by a single exponential at low (less than 10(-6) M) [Ca]i but a component of fast openings (to less than 1.0 ms) was observed at all potentials at [Ca]i 2.5 mM. Closed time distribution required the sum of three exponentials to fit it all [Ca]i greater than 10(-7) M; at [Ca]i 10(-6) M or greater evidence of a fourth component, probably caused by Ca block of open channels, was obtained. Raising [Ca]i increased the mean duration of the (long) open state and decreased or had no effect on the duration of short, intermediate, and long mean closed states.

O-160 - Ca2+ release from dispersed single smooth muscle cells

O-160 - Ca2+ release from dispersed single smooth muscle cells