Contraction of smooth muscle is initiated, and to a lesser extent maintained, by a rise in the concentration of free calcium in the cell cytoplasm ([Ca 2+ ] i ). This activator calcium can originate from two intimately linked sources ± the extracellular space and intracellular stores, most notably the sarcoplasmic reticulum. Smooth muscle contraction activated by excitatory neurotransmitters or hormones usually involves a combination of calcium release and calcium entry. The latter occurs through a variety of calcium permeable ion channels in the sarcolemma membrane. The bestcharacterized calcium entry pathway utilizes voltage-operated calcium channels (VOCCs). However, also present are several types of calcium-permeable channels which are non-voltage-gated, including the so-called receptor-operated calcium channels (ROCCs), activated by agonists acting on a range of G-protein-coupled receptors, and store-operated calcium channels (SOCCs), activated by depletion of the calcium stores within the sarcoplasmic reticulum. In this article we will review the electrophysiological, functional and pharmacological properties of ROCCs and SOCCs in smooth muscle and highlight emerging evidence that suggests that the two channel types may be closely related, being formed from proteins of the Transient Receptor Potential Channel (TRPC) family.

1 By use of the whole-cell configuration of the patch-clamp technique, membrane currents induced by cyclopiazonic acid (CPA; an inhibitor of the sarcoplasmic reticulum (SR) calcium-ATPase) were investigated in single smooth muscle cells freshly dispersed from the mouse anococcygeus. Voltagedependent calcium currents were blocked with extracellular nifedipine and caesium and tetraethylammonium chloride were used to block voltage-dependent potassium currents. 2 At a holding potential of -40 mV, CPA (10 tiM) activated an inward current that consisted of two distinct components. The first was an initial transient current with an amplitude of 19.6 + 1.9 pA while the second was sustained and had an amplitude of 3.5 + 0.3 pA. 3 The current-voltage (I-V) relationship for the transient current showed marked outward rectification. The current had a reversal potential of 9.1 + 1.1 mV which was shifted to 29.0 + 4.2 mV when the extracellular chloride concentration was lowered from 148.4 to 58.4 mM. The sustained current had a near-linear I-V relationship and a reversal potential of 31.0+2.7 mV. Removal of extracellular calcium had no effect on the transient current, but shifted the reversal potential of the sustained current to 18.2+ 5.7 mV. 3 The initial transient current was abolished in cells bathed in extracellular solutions containing the chloride channel blockers, 4,4' diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS; 1 mM) or anthracene-9-carboxylic acid (A-9-C; 1 mM), and was absent in cells containing the calcium buffers EGTA (1 to 5 mM) or BAPTA (10 mM). The second sustained current was unaffected by either the chloride channel blockers or the intracellular calcium buffers. 4 Treatment of the cells with caffeine (10 mM) produced similar inward currents to those produced by CPA. In the presence of caffeine, CPA (10 gM) induced no further inward current. 5 In organ bath studies, CPA (10 gM)-induced contractions of the mouse anococcygeus were inhibited by cadmium and nickel (both 50-400 uM) and the general calcium entry blocker, SKF 96365 (10 gM); lanthanum and gadolinium had no effect at concentrations up to 400 gM. The pharmacology of the CPA-induced non-selective cation current mirrored that of the CPA-induced whole muscle contraction being reversed by cadmium (100 tM) and SKF 96365 (10 uM), but unaffected by lanthanum (400 gM).The initial chloride conductance was unaffected by cadmium, SKF 96365 or lanthanum. 6 It is concluded that CPA activates a transient calcium-dependent chloride current as a consequence of calcium release from intracellular stores; this current would result in depolarization and opening of voltage-operated calcium channels, which mediate the nifedipine-sensitive component of muscle contraction. In addition, as a result of emptying the SR, CPA activates a non-selective cation conductance which may underlie the nifedipine-insensitive calcium entry process utilised during sustained contraction.

No abstract

Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 h depending on the experimental design and yields 16-33 cell recordings.

during Ca2' re-loading, increased the subsequent residual contraction to carbachol in Ca2+-free medium.7 At higher concentrations, SNP (0.1-10 fM) produced a partial relaxation of the sustained contraction to U46619 in Ca2+-free medium. 8 Thus, the relaxant potency of the nitrergic stimuli was dependent on the agent and mechanism used to induce tone in the preparation. Examination of the contractile/relaxant interactions suggests that altered Ca2+ sequestration and inhibition of contractile protein function may underlie nitrergic relaxations of this tissue.

1 The effects of sodium nitroprusside (SNP) on the non-selective cation current activated in response to intracellular calcium store depletion were studied using the whole-cell patch-clamp technique in single smooth muscle cells isolated from the mouse anococcygeus. Voltage-dependent calcium currents were blocked with extracellular nifedipine, and caesium and tetraethylammonium chloride were used to block voltage-dependent potassium currents. Calcium stores were depleted with caffeine (10 mM), carbachol (50 pM) or cyclopiazonic acid (CPA 10 pM; an inhibitor of the sarcoplasmic reticulum [SR] calciumATPase). 2 At a holding potential of -40 mV, both CPA and caffeine activated inward currents which consisted of two clearly distinguishable components; an initial transient current followed by a smaller sustained current. In the case of CPA, the amplitudes of the transient and sustained components were 19.7 + 2.1 pA and 3.5 + 0.3 pA respectively, whilst the equivalent values for caffeine were 188 + 21 and 4.8 + 0.3 pA. As described previously, the transient current results from activation of a calciumdependent chloride conductance whilst the sustained current is a non-selective cation current, activated following intracellular calcium store depletion. 3 The muscarinic receptor agonist, carbachol, also activated a transient followed by a sustained current with amplitudes of 238 + 55 and 4.7 + 0.5 pA respectively. Superimposed on the sustained current were regular, oscillations of calcium-activated chloride current. 4 Both the transient and the sustained currents activated by CPA were absent in cells pretreated with SNP (10 gM). Application of SNP to a cell following activation of the sustained current by CPA inhibited the current by 88.6 ± 3.8%. SNP (10 pM) did not inhibit the transient current activated by caffeine but abolished the sustained current.5 SNP (10 gM) had no effect on the initial transient current activated by carbachol (50 gM). However, it did inhibit the oscillations in the inward current. In recordings from cells bathed in extracellular solution containing the chloride channel blocker, anthracene-9-carboxylic acid (A-9-C; 1 mM), carbachol activated only a sustained current. This current was inhibited by 88.1+ 6.5% by a concomitant application of SNP (10 gM) and was absent in cells pretreated with the nitrovasodilator.6 The effects of SNP on the currents activated by caffeine (10 mM) were mimicked by 8-bromo-cycic GMP (200 Mm); thus the nucleotide had no effect on the transient current activated by caffeine but abolished the sustained current. The effects of SNP, but not those of 8-bromo-cyclic GMP, were inhibited by the nitric oxide-sensitive guanylyl cyclase inhibitor, 1H-[1, 2, 4]oxadiazolo[4, 3-a]quinoxaline-i-one (ODQ; 1 Mm). ODQ alone produced a significant increase in the size of the sustained current activated by caffeine (7.8 + 0.7 pA). 7 These findings suggest that SNP activates guanylyl cyclase to inhibit the non-selective cation current activated as a result of intracellular calcium store d...

Abstract-Adrenomedullin (AM) levels are elevated in cardiovascular disease, but little is known of the role of specific receptor components. AM acts via the calcitonin receptor-like receptor (CLR) interacting with a receptor-activitymodifying protein (RAMP). The AM 1 receptor is composed of CLR and RAMP2, and the calcitonin gene-related peptide (CGRP) receptor of CLR and RAMP1, as determined by molecular and cell-based analysis. This study examines the relevance of RAMP2 in vivo. Transgenic (TG) mice that overexpress RAMP2 in smooth muscle were generated. The role of RAMP2 in the regulation of blood pressure and in vascular function was investigated. Basal blood pressure, acute angiotensin II-raised blood pressure, and cardiovascular properties were similar in wild-type (WT) and TG mice. However, the hypotensive effect of IV AM, unlike CGRP, was enhanced in TG mice (PϽ0.05), whereas a negative inotropic action was excluded by left-ventricular pressure-volume analysis. Plasma concentrations are raised in several cardiovascular conditions including heart failure and sepsis. 4 -6 Despite the possible importance of AM in cardiovascular biology, little is known of the receptor components involved. Their influence in vivo has only been inferred from molecular and cellular studies. 7,8 AM is a member of the calcitonin family of peptides and acts via unique G protein-linked receptors, composed of a common calcitonin receptor-like receptor (CLR) associated with 1 of 3 receptor-activity-modifying proteins (RAMPs). The complex of CLR with RAMP1 defines a CGRP receptor, whereas CLR/RAMP2 is an AM 1 receptor and the CLR/ RAMP3 heterodimer is known as an AM 2 receptor. 9,10 Studies in a range of species show that CGRP is more potent as a vasodilator than AM and that AM is an agonist at both AM and CGRP receptors, whereas CGRP predominantly acts via CGRP receptors. Both receptors are primarily linked to cAMP synthesis, 8,11 but vascular relaxation can also occur via endothelial-derived NO-dependent mechanisms. 12 Experiments in mice have demonstrated the importance of AM in vascular development as homozygous AM knockout mice exhibit a fatal phenotype. 13,14 However, AM transgenics and heterozygote knockouts show that AM plays a protective role in sepsis, 15 ischemia, 16 and cardiovascular damage. 17 Thus upregulation of the peptide correlates with important cardiovascular activities. Recently, a novel form of AM, AM2, has been identified that possesses vasodilator activity. 18 Currently, little is known of the functional importance of receptor

The properties of the calcium stores coupled to a depletion-operated cation current (IDOC) proposed to underlie capacitative calcium entry were studied in single smooth muscle cells isolated from the mouse anococcygeus using the whole-cell patch-clamp technique. Both caffeine (10 mM) and carbachol (50 microM) activated an initial, large ( approximately 200 pA), transient, calcium-dependent chloride current (IClCa) followed by a smaller ( approximately 10 pA) sustained, non-selective cation current (IDOC). Intracellular application of heparin (5 mg ml-1) abolished the response to carbachol but potentiated that to caffeine. Ryanodine (3 microM) activated IDOC but not IClCa; ryanodine (30 microM) failed to produce any response. Both concentrations of ryanodine abolished the response to caffeine and prevented activation of IClCa by carbachol. In the presence of 30 microM, but not 3 microM, ryanodine, carbachol was able to activate IDOC. Cyclopiazonic acid (CPA; 10 microM) abolished the response to carbachol; however, caffeine was still able to activate IClCa. In whole-muscle tension recordings, ryanodine at both 3 and 30 microM produced contractions of the tissue but only that in response to the lower concentration was maintained. Thus, depletion of either inositol 1,4, 5-trisphosphate-(IP3-) sensitive or ryanodine-sensitive calcium stores is able to activate IDOC, and, by extension, capacitative calcium entry in this tissue.