Functional properties and pharmacological inhibition of ASIC channels in the human SJ-RH30 skeletal muscle cell line.

The rhabdomyosarcoma cell line (SJ-RH30) exhibits both ultrastructural and electrophysiological hallmarks of mammalian skeletal muscle. We have used patch-clamp electrophysiology to study acid-gated currents in these cells. At a holding potential of -60 mV, rapid application of extracellular solutions of pH 6.5 produced inward current responses in approximately 85% of cells. The amplitude of these responses exhibited a marked pH dependence. In addition, the kinetics of both activation and desensitization were faster at more acidic pH, whereas the deactivation rate was pH independent. Repeated applications of a pH 6.0 solution produced a tachyphalaxis that could be substantially attenuated by reducing the duration of the acid challenge and increasing the interstimulus interval. The current-voltage relationship of the acid-induced currents was linear at positive potentials but an area of negative slope conductance was observed in the negative potential range. This was not eliminated by removal of extracellular Ca(2+), a manoeuvre which did, however, substantially increase current amplitude. Changing the transmembrane Na(+) gradient altered the current-voltage relationship in a fashion commensurate with an underlying conductance predominantly permeable to Na(+). Pharmacologically, acid-induced currents were blocked 84.4 +/- 1.2% by 30 microm amiloride and 91.8 +/- 3.0% by a 1 : 1000 dilution of Psalmopoeus cambridgei venom. Inhibition by both agents could be reversed by a short period of compound washout. By contrast, APETx2 had no significant effect on acid-evoked currents. These observations strongly suggest the acid-induced current is mediated by ASIC1 channels. In agreement with this, current responses of SJ-RH30 cells to a pH 6.0 challenge were greatly enhanced by extracellular lactate. These data demonstrate the presence of ASIC1 channels in a cell line with skeletal muscle characteristics. In addition, significant levels of ASIC1 and ASIC3 mRNA were found in skeletal muscle tissue samples. These findings are discussed with regard to the role such a conductance might play if present in skeletal muscle in vivo.

Does the sunset of mental health parity really matter?

The 1996 Mental Health Parity Act (MHPA), which became effective in January 1998, is scheduled to expire in September 2001. This article provides an overview of what the MHPA intended to do and what it actually has accomplished. We summarize state legislature actions through the end of 2000 and report on their effects on employer-sponsored mental health coverage using a national survey fielded in 1999-2000. We then discuss possible amendments to the MHPA and reforms beyond full parity that might be considered.

Toward full mental health parity and beyond.

The 1996 Mental Health Parity Act (MHPA), which became effective in January 1998, is scheduled to expire in September 2001. This paper examines what the MHPA accomplished and steps toward more comprehensive parity. We explain the strategic and self-reinforcing link of parity with managed behavioral health care and conclude that the current path will be difficult to reverse. The paper ends with a discussion of what might be behind the claims that full parity in mental health benefits is insufficient to achieve true equity and whether additional steps beyond full parity appear realistic or even desirable.

Nerve evoked P2X receptor contractions of rat mesenteric arteries; dependence on vessel size and lack of role of L-type calcium channels and calcium induced calcium release.

1. Contractile responses to short trains of nerve stimulation have been characterized in small, medium and large arteries from the rat mesenteric circulation (5th - 6th, 2nd - 3rd and 1st order, respectively). In addition, sources of calcium for smooth muscle contraction have been investigated. 2. Nerve stimulation (10 pulses at 10 Hz) evoked reproducible contractions. The P2 receptor antagonist suramin (100 microM) reduced constrictions by 65.3+/-7.4, 82.7+/-3.3 and 3.1+/-6.1% in small, medium and large arteries respectively. The alpha-adrenoceptor antagonist prazosin (0.1 microM) reduced responses by 32.6+/-2.6, 27.0+/-1.5 and 97.0+/-1.9% respectively. 3. The L-type calcium channel antagonist nifedipine (1 microM) reduced nerve-evoked contractions by 2.8+/-3.3, 10.0+/-3.7 and 13.5+/-2.7% in small, medium and large arteries respectively. When the adrenergic component of contraction was blocked by prazosin (0.1 microM) nifedipine reduced responses by 4.6+/-7.9, 14.3+/-2.0 and 3.0+/-1.9% respectively. Contractile responses to exogenous alpha,beta-meATP were unaffected by the depletion of calcium stores with cyclopiazonic acid (30 microM). This indicates that mobilization of calcium from internal stores is not required for P2X receptor mediated smooth muscle contraction. We conclude that for neurogenic responses, the P2X receptor mediated component of constriction dominates in small mesenteric arteries (3rd -- 6th order) while in large arteries (1st order) noradrenaline mediates contraction. For P2X receptor mediated responses all the calcium required for smooth muscle contraction enters the cell directly through P2X receptor channels.

Effects of diadenosine polyphosphates (Ap(n)As) and adenosine polyphospho guanosines (Ap(n)Gs) on rat mesenteric artery P2X receptor ion channels.

Diadenosine polyphosphates (Ap(n)As, n=3 - 7) and adenosine polyphospho guanosines (Ap(n)Gs, n=3 - 6) are naturally occurring vasoconstrictor substances found in platelets. These vasoconstrictor actions are thought to be mediated through the activation of P2X receptors for ATP. The effects of Ap(n)As and Ap(n)Gs at P2X receptors on rat mesenteric arteries were determined in contraction studies and using the patch clamp technique on acutely dissociated artery smooth muscle cells. P2X(1) receptor immunoreactivity was detected in the smooth muscle layer of artery rings. The sensitivity to alpha,beta-methylene ATP and desensitizing nature of rat mesenteric artery P2X receptors correspond closely to those of recombinant P2X(1) receptors. Ap(4)A, Ap(5)A and Ap(6)A evoked concentration dependent P2X receptor inward currents which desensitized during the application of higher concentrations of agonist. The agonist order of potency was Ap(5)A> or = Ap(6)A> or = Ap(4)A >> Ap(3)A. Ap(2)A and Ap(7)A were ineffective. Similar results were obtained in contraction studies except for Ap(7)A which evoked a substantial contraction. Ap(n)Gs (n=2 - 6)(30 microM) evoked P2X receptor inward currents in mesenteric artery smooth muscle cells. Ap(n)Gs (n=4 - 6) were less effective than the corresponding Ap(n)A. This study shows that at physiologically relevant concentrations Ap(n)As and Ap(n)Gs can mediate contraction of rat mesenteric arteries through the activation of P2X(1)-like receptors. However the activity of the longer chain polyphosphates (n=6 - 7) may be overestimated in whole tissue studies due to metabolic breakdown to yield the P2X receptor agonists ATP and adenosine tetraphosphate. British Journal of Pharmacology (2000) 129, 124 - 130

Reduced vas deferens contraction and male infertility in mice lacking P2X1 receptors.

P2X1 receptors for ATP are ligand-gated cation channels, present on many excitable cells including vas deferens smooth muscle cells. A substantial component of the contractile response of the vas deferens to sympathetic nerve stimulation, which propels sperm into the ejaculate, is mediated through P2X receptors. Here we show that male fertility is reduced by approximately 90% in mice with a targeted deletion of the P2X1 receptor gene. Male mice copulate normally--reduced fertility results from a reduction of sperm in the ejaculate and not from sperm dysfunction. Female mice and heterozygote mice are unaffected. In P2X1-receptor-deficient mice, contraction of the vas deferens to sympathetic nerve stimulation is reduced by up to 60% and responses to P2X receptor agonists are abolished. These results show that P2X1 receptors are essential for normal male reproductive function and suggest that the development of selective P2X1 receptor antagonists may provide an effective non-hormonal male contraceptive pill. Also, agents that potentiate the actions of ATP at P2X1 receptors may be useful in the treatment of male infertility.

Properties of P2X and P2Y receptors are dependent on artery diameter in the rat mesenteric bed.

P2 receptor mediated contractile responses have been characterized in different diameter arteries from the rat mesenteric arterial vasculature (first, second to third and fifth to sixth order for large, medium and small arteries) using wire myograph and diamtrak video imaging. alpha,ss-methylene ATP (alpha,beta-meATP) evoked transient concentration-dependent contractions in mesenteric arteries with EC(50) values of 0.4, 2.5 and 107 microM for small, medium and large arteries respectively. Suramin (10 - 100 microM) produced substantial parallel rightward shifts of the concentration-response curve to alpha,beta-meATP in small and medium-sized arteries with pA(2) of 5.1. Responses in large vessels were unaffected by suramin. Immunohistochemical analysis of arterial sections revealed no substantial differences in expression patterns of P2X receptors between different sizes of artery. P2X(1) receptors were expressed at high levels, P2X(4) and P2X(5) receptors were also detected on smooth muscle. The P2X receptor response is dominated by P2X(1) receptor in small and medium arteries but the nature of the receptor mediating the suramin insensitive alpha,beta-meATP mediated response in large arteries is unclear. The P2Y receptor agonist UTP was significantly more potent in small than in medium or large arteries (EC(50) values: 15.0 microM small, 88.5 microM diamtrak medium 1.6 mM myography medium and 1.4 mM large). Responses in both small and medium-sized vessels were reduced by suramin (30 - 100 microM). The sensitivity to UTP and suramin indicates the presence of P2Y(2) receptors. This study shows that P2 receptors do not have a homogenous phenotype throughout the mesenteric vascular bed and that the properties depend on artery size.