Kv1.5 is a major component underlying the A-type potassium current in retinal arteriolar smooth muscle.

Little is known about the molecular characteristics of the voltage-activated K(+) (K(v)) channels that underlie the A-type K(+) current in vascular smooth muscle cells of the systemic circulation. We investigated the molecular identity of the A-type K(+) current in retinal arteriolar myocytes using patch-clamp techniques, RT-PCR, immunohistochemistry, and neutralizing antibody studies. The A-type K(+) current was resistant to the actions of specific inhibitors for K(v)3 and K(v)4 channels but was blocked by the K(v)1 antagonist correolide. No effects were observed with pharmacological agents against K(v)1.1/2/3/6 and 7 channels, but the current was partially blocked by riluzole, a K(v)1.4 and K(v)1.5 inhibitor. The current was not altered by the removal of extracellular K(+) but was abolished by flecainide, indicative of K(v)1.5 rather than K(v)1.4 channels. Transcripts encoding K(v)1.5 and not K(v)1.4 were identified in freshly isolated retinal arterioles. Immunofluorescence labeling confirmed a lack of K(v)1.4 expression and revealed K(v)1.5 to be localized to the plasma membrane of the arteriolar smooth muscle cells. Anti-K(v)1.5 antibody applied intracellularly inhibited the A-type K(+) current, whereas anti-K(v)1.4 antibody had no effect. Co-expression of K(v)1.5 with K(v)beta1 or K(v)beta3 accessory subunits is known to transform K(v)1.5 currents from delayed rectifers into A-type currents. K(v)beta1 mRNA expression was detected in retinal arterioles, but K(v)beta3 was not observed. K(v)beta1 immunofluorescence was detected on the plasma membrane of retinal arteriolar myocytes. The findings of this study suggest that K(v)1.5, most likely co-assembled with K(v)beta1 subunits, comprises a major component underlying the A-type K(+) current in retinal arteriolar smooth muscle cells

Evidence for two endothelin Et(A) receptor subtypes in rabbit arteriolar smooth muscle.

1. Effects of endothelin-1 (Et-1) were studied on membrane currents in choroidal arteriolar smooth muscle by using perforated patch-clamp recordings. 2. Et-1 (10 nM) activated oscillatory Ca(2+)-activated Cl(-)-currents (I(Cl(Ca))) which could not be reversed by washing out. 3. Currents through L-type Ca(2+) channels were resolved in a divalent free medium (I(Ca(L)Na)). Et-1 reduced I(Ca(L)Na) by 75 +/- 7% within 30 s and this effect faded over 5 min, when the depression remained constant. On washing out Et-1, I(Ca(L)Na) almost completely recovered within 10 s. 4. BQ123 (1 microM), a peptide Et(A) receptor blocker, prevented the activation of I(Cl(Ca)), but failed to inhibit I(Cl(Ca)) transients once they had been initiated. In contrast, BQ123 not only prevented but also reversed the inhibition of I(Ca(L)Na) by Et-1. BQ788 (1 microM), an Et(B) receptor antagonist, did not prevent the activation of I(Cl(Ca)) or the inhibition of I(Ca(L)Na) by Et-1. 5. ABT-627 (10 nM), a non-peptide Et(A) receptor antagonist also blocked the activation of I(Cl(Ca)). However, on I(Ca(L)Na), ABT-627 (10 nM) mimicked the action of Et-1 an effect blocked by BQ123 suggesting that ABT-627 acted as an agonist. 6. The data are consistent with choroidal arteriolar smooth muscle cells having two types of Et(A) receptor, one where BQ123 is an antagonist and ABT-627 an agonist, where ligands dissociate freely and this receptor is coupled to inhibition of L-type Ca(2+) channels. In the other, BQ123 and ABT-627 are both antagonists and with Et-1 the receptor converts to a high affinity state producing the classical irreversible activation I(Cl(Ca)).

The smooth muscle pharmacology of maximakinin, a receptor-selective, bradykinin-related nonadecapeptide from the venom of the Chinese toad, Bombina maxima.

Structural homologues of vertebrate regulatory peptides found in defensive skin secretions of anuran amphibians often display enhanced bioactivity and receptor binding when compared with endogenous mammalian peptide ligands. Maximakinin, a novel N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the skin venom of a Chinese toad (Bombina maxima), displays such activity enhancement when compared with bradykinin but is additionally highly selective for mammalian arterial smooth muscle bradykinin receptors displaying a 50-fold increase in molar potency in this smooth muscle type. In contrast, a 100-fold decrease in molar potency was observed at bradykinin receptors in intestinal and uterine smooth muscle preparations. Maximakinin has thus evolved as a “smart” defensive weapon in the toad with receptor/tissue selective targeting. Natural selection of amphibian skin venom peptides for antipredator defence, through inter-species delivery by an exogenous secretory mode, produces subtle structural stabilisation modifications that can potentially provide new insights for the design of selectively targeted peptide therapeutics.

Kit-positive interstitial cells in the rabbit urethra: structural relationships with nerves and smooth muscle.

OBJECTIVE: To identify interstitial cells (ICs) in the wall of the rabbit urethra using antibodies to the Kit receptor, and to examine their location, morphology and relationship with nerves and smooth muscle cells (SMCs), as studies of enzymatically isolated cells from the rabbit urethra have established that there are specialized cells that show spontaneous electrical activity and have morphological properties of ICs. MATERIALS AND METHODS: Urethral tissues from rabbits were fixed, labelled with antibodies and examined with confocal microscopy. Some specimens were embedded in paraffin wax and processed for histology. Histological sections from the most proximal third and mid-third region of rabbit urethra were stained with Masson's Trichrome to show their cellular arrangement. RESULTS: Sections from both regions had outer longitudinal and inner circular layers of SM, and a lamina propria containing connective tissue and blood vessels; the lumen was lined with urothelial cells. The mid-third region had a more developed circular SM layer than the most-proximal samples, and had extensive inner longitudinal SM bundles in the lamina propria. Labelling with anti-Kit revealed immunopositive cells within the wall of the rabbit urethra, in the circular and longitudinal layers of the muscularis. Double-labelling with an antibody to SM myosin showed Kit-positive cells on the boundary of the SM bundles, orientated parallel to the axis of the bundles. Others were in spaces between the bundles and often made contact with each other. Kit-positive cells were either elongated, with several lateral branches, or stellate with branches coming from a central soma. Similar cells could be labelled with vimentin antibodies. Their relationship with intramural nerves was examined by double immunostaining with an anti-neurofilament antibody. There were frequent points of contact between Kit-positive cells and nerves, with similar findings in specimens double-immunostained with anti-neuronal nitric oxide synthase (nNOS). CONCLUSION: Kit-positive ICs were found within the SM layers of the rabbit urethra, in association with nerves, on the edge of SM bundles and in the interbundle spaces. The contact with nNOS-containing neurones might imply participation in the nitrergic inhibitory neurotransmission of the urethra. PMID: 17212607 [PubMed - indexed for MEDLINE]

Cellular physiology of retinal and choroidal arteriolar smooth muscle cells.

Control of ocular blood flow occurs predominantly at the level of the retinal and choroidal arterioles. The present article provides an overview of the Ca2 + handling mechanisms and plasmalemmal ion channels involved in the regulation of retinal and choroidal arteriolar smooth muscle tone. Increases in global intracellular free Ca2 + ([Ca2 +]i) involve multiple mechanisms, including agonist-dependent release of Ca2 + from intracellular stores through activation of the inositol trisphosphate (IP3) pathway. Ca2 + enters by voltage-dependent L-type Ca2 + channels and novel dihydropyridine-sensitive store-operated nonselective cation channels. Ca2 + extrusion is mediated by plasmalemmal Ca2 +-ATPases and through Na+/Ca2+ exchange. Local Ca2 + transients (Ca2 + sparks) play an important excitatory role, acting as the building blocks for more global Ca2 + signals that can initiate vasoconstriction. K+ and Cl- channels may also affect cell function by modulating membrane potential. The precise contribution of each of these mechanisms to the regulation of retinal and choroidal perfusion in vivo warrants future investigation.

Modification of smooth muscle Ca2+-sparks by tetracaine: evidence for sequential RyR activation.

Spontaneous Ca(2+)-sparks were imaged using confocal line scans of fluo-4 loaded myocytes in retinal arterioles. Tetracaine produced concentration-dependent decreases in spark frequency, and modified the spatiotemporal characteristics of residual sparks. Tetracaine (10 microM) reduced the rate of rise but prolonged the average rise time so that average spark amplitude was unaltered. The mean half-time of spark decay was also unaffected, suggesting that spark termination, although delayed, remained well synchronized. Sparks spread transversely across the myocytes in these vessels, and the speed of spread within individual sparks was slowed by approximately 60% in 10 microM tetracaine, as expected if the spark was propagated across the cell but the average P(o) for RyRs was reduced. Staining of isolated vessels with BODIPY-ryanodine and di-4-ANEPPS showed that RyRs were located both peripherally, adjacent to the plasma membrane, and in transverse extensions of the SR from one side of the cell to the other. Immuno-labelling of retinal flat mounts demonstrated the presence RyR(2) in arteriole smooth muscle but not RyR(1). We conclude that Ca(2+)-sparks in smooth muscle can result from sequential activation of RyRs distributed over an area of several microm(2), rather than from tightly clustered channels as in striated muscle.

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.