Role of calcium influx through voltage-operated calcium channels and of calcium mobilization in the physiology of Schistosoma mansoni muscle contractions

We tested the hypothesis that voltage-operated Ca2+ channels mediate an extracellular Ca2+ influx in muscle fibres from the human parasite Schistosoma mansoni and, along with Ca2+ mobilization from the sarcoplasmic reticulum, contribute to Muscle contraction. Indeed, whole-cell voltage clamp revealed voltage-gated inward currents carried by divalent ions with a peak current elicited by steps to + 20 mV (from a holding potential of -70 mV). Depolarization of the fibres by elevated extracellular K+ elicited contractions that were completely dependent on extracellular Ca2+ and inhibited by nicardipine (half inhibition at 4(.)1 mu M). However these contractions were not very sensitive to other classical blockers of voltage-gated Ca2+ channels, indicating that the schistosome Muscle channels have an atypical pharmacology when compared to their mammalian counterparts. Furthermore, the contraction induced by 5 mM caffeine was inhibited after depletion of the sarcoplasmic reticulum either with thapsigargin (10 mu M) or ryanodine (10 mu M). These data suggest that voltage-operated Ca2+ channels docontribute to S. mansoni contraction as does the mobilization of stored Ca2+, despite the small volume of sarcoplasmic reticulum in schistosome smooth muscles.

Ca2+-activated Cl- current in retinal arteriolar smooth muscle.

PURPOSE: To characterize the biophysical, pharmacologic, and functional properties of the Ca(2+)-activated Cl(-) current in retinal arteriolar myocytes. METHODS: Whole-cell perforated patch-clamp recordings were made from myocytes within intact isolated arteriolar segments. Arteriolar tone was assessed using pressure myography. RESULTS: Depolarizing of voltage steps to -40 mV and greater activated an L-type Ca(2+) current (I(Ca(L))) that was followed by a sustained current. Large tail currents (I(tail)) were observed on stepping back to -80 mV. The sustained current and I(tail) reversed close to 0 mV in symmetrical Cl(-) concentrations. The ion selectivity sequence for I(tail) was I(-)> Cl(-)> glucuronate. Outward I(tail) was sensitive to the Cl(-) channel blockers 9-anthracene-carboxylic acid (9-AC; 1 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS; 1 mM), and disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS; 1 mM), but only DIDS produced a substantial (78%) block of inward tail currents at -100 mV. I(tail) was decreased in magnitude when the normal bathing medium was substituted with Ca(2+)-free solution or if I(Ca(L)) was inhibited by 1 microM nimodipine. Caffeine (10 mM) produced large transient currents that reversed close to the Cl(-) equilibrium potential and were blocked by 1 mM DIDS or 100 microM tetracaine. DIDS had no effect on basal vascular tone in pressurized arterioles but dramatically reduced the level of vasoconstriction observed in the presence of 10 nM endothelin-1. CONCLUSIONS: Retinal arteriolar myocytes have I(Cl(Ca)), which may be activated by Ca(2+) entry through L-type Ca(2+) channels or Ca(2+) release from intracellular stores. This current appears to contribute to agonist-induced retinal vasoconstriction.

Deletion of TRPC4 and TRPC6 in Mice Impairs Smooth Muscle Contraction and Intestinal Motility In Vivo.

BACKGROUND & AIMS: Downstream effects of muscarinic receptor stimulation in intestinal smooth muscle include contraction and intestinal transit. We thought to determine whether classic transient receptor potential (TRPC) channels integrate the intracellular signaling cascades evoked by the stimulated receptors and thereby contribute to the control of the membrane potential, Ca-influx, and cell responses. METHODS: We created trpc4-, trpc6-, and trpc4/trpc6-gene-deficient mice and analyzed them for intestinal smooth muscle function in vitro and in vivo. RESULTS: In intestinal smooth muscle cells TRPC4 forms a 55 pS cation channel and underlies more than 80% of the muscarinic receptor-induced cation current (mI(CAT)). The residual mI(CAT) depends on the expression of TRPC6, indicating that TRPC6 and TRPC4 determine mI(CAT) channel activity independent of other channel subunits. In TRPC4-deficient ileal myocytes the carbachol-induced membrane depolarizations are diminished greatly and the atropine-sensitive contraction elicited by acetylcholine release from excitatory motor neurons is reduced greatly. Additional deletion of TRPC6 aggravates these effects. Intestinal transit is slowed down in mice lacking TRPC4 and TRPC6. CONCLUSIONS: In intestinal smooth muscle cells TRPC4 and TRPC6 channels are gated by muscarinic receptors and are responsible for mI(CAT). They couple muscarinic receptors to depolarization of intestinal smooth muscle cells and voltage-activated Ca(2+)-influx and contraction, and thereby accelerate small intestinal motility in vivo.

Amolopkinins W1 and W2 – novel bradykinin-related peptides (BRPs) from the skin of the Chinese torrent frog, Amolops wuyiensis: antagonists of bradykinin-induced smooth muscle contraction of the rat ileum.

Bradykinin-related peptides (BRPs) represent one of the most widespread and closely studied families of amphibian defensive skin secretion peptides. Apart from canonical bradykinin (RPPGFSPFR) that was first reported in skin extracts of the European brown frog, Rana temporaria, many additional site-substituted, N- and/or C-terminally extended peptides have been isolated from skin extracts and secretions from representative species of the families Ranidae, Hylidae, Bombinatoridae and Leiopelmatidae. The most diverse range of BRPs has been found in ranid frog skin secretions and this probably reflects the diversity and number of species studied and their associated life histories within this taxon. Amolops (torrent or cascade frogs) is a genus within the Ranidae that has been poorly studied. Here we report the presence of two novel BRPs in the skin secretions of the Chinese Wuyi Mountain torrent frog (Amolops wuyiensis). Amolopkinins W1 and W2 are dodecapeptides differing in only one amino acid residue at position 2 (Val/Ala) that are essentially (Leu1, Thr6)-bradykinins extended at the N-terminus by either RVAL (W1) or RAAL (W2). Amolopkinins W1 and W2 are structurally similar to amolopkinin L1 from Amolops loloensis and the major BRP (Leu1, Thr6, Trp8)-bradykinin from the skin of the Japanese frog, Rana sakuraii. A. wuyiensis amolopkinins were separately encoded as single copies within discrete precursors of 61 amino acid residues as deduced from cloned skin cDNA. Synthetic replicates of both peptides were found to potently antagonize the contractile effects of canonical bradykinin on isolated rat ileum smooth muscle preparations. Amolopkinins thus appear to represent a novel sub-family of ranid frog skin secretion BRPs.

Kassina senegalensis skin tachykinins: Molecular cloning of kassinin and (Thr2, Ile9)-kassinin biosynthetic precursor cDNAs and comparative bioactivity of mature tachykinins on the smooth muscle of rat urinary bladder

Tachykinins are among the most widely-studied families of regulatory peptides characterized by a highly-conserved C-terminal -Phe-X-Gly-Leu-Met.amide motif, which also constitutes the essential bioactive core. The amphibian skin has proved to be a rich source of these peptides with physalaemin from the skin of Physalaemus fuscomaculatus representing the archetypal aromatic tachykinin (X = Tyr or Phe) and kassinin from the skin of Kassina senegalensis representing the archetypal aliphatic tachykinin in which X = Val or Ile. Despite the primary structures of both mature peptides having been known for at least 30 years, neither the structures nor organizations of their biosynthetic precursors have been reported. Here we report the structure and organization of the biosynthetic precursor of kassinin deduced from cDNA cloned from a skin secretion library. In addition, a second precursor cDNA encoding the novel kassinin analog (Thr2, Ile9)-kassinin was identified as was the predicted mature peptide in skin secretion. Both transcripts exhibited a high degree of nucleotide sequence similarity and of open-reading frame translated amino acid sequences of putative precursor proteins. The translated preprotachykinins each consisted of 80 amino acid residues encoding single copies of either kassinin or its site-substituted analog. Synthetic replicates of each kassinin were found to be active on rat urinary bladder smooth muscle at nanomolar concentrations. The structural organization of both preprotachykinins differs from that previously reported for those of Odorrana grahami skin indicating a spectrum of diversity akin to that established for amphibian skin preprobradykinins.