Rat Tail Artery Smooth Muscle Research Articles

BK Channels in Tail Artery Vascular Smooth Muscle Cells of Normotensive (WKY) and Hypertensive (SHR) Rats Possess Similar Calcium Sensitivity But Different Responses to the Vasodilator Iloprost.

It has been reported that, in the spontaneously hypertensive rat (SHR) model of hypertension, different components of the G-protein/adenylate cyclase (AC)/Calcium-activated potassium channel of high conductance (BK) channel signaling pathway are altered differently. In the upstream part of the pathway (G-protein/AC), a comparatively low efficacy has been established, whereas downstream BK currents seem to be increased. Thus, the overall performance of this signaling pathway in SHR is elusive. For a better understanding, we focused on one aspect, the direct targeting of the BK channel by the G-protein/AC pathway and tested the hypothesis that the comparatively low AC pathway efficacy in SHR results in a reduced agonist-induced stimulation of BK currents. This hypothesis was investigated using freshly isolated smooth muscle cells from WKY and SHR rat tail artery and the patch-clamp technique. It was observed that: (1) single BK channels have similar current-voltage relationships, voltage-dependence and calcium sensitivity; (2) BK currents in cells with a strong buffering of the BK channel activator calcium have similar current-voltage relationships; (3) the iloprost-induced concentration-dependent increase of the BK current is larger in WKY compared to SHR; (4) the effects of activators of the PKA pathway, the catalytic subunit of PKA and the potent and selective cAMP-analogue Sp-5,6-DCl-cBIMPS on BK currents are similar. Thus, our data suggest that the lower iloprost-induced stimulation of the BK current in freshly isolated rat tail artery smooth muscle cells from SHR compared with WKY is due to the lower efficacy of upstream elements of the G-Protein/AC/BK channel pathway.

Pharmacological Effects of a Novel Bradykinin-Related Peptide (RR-18) from the Skin Secretion of the Hejiang Frog (Ordorrana hejiangensis) on Smooth Muscle.

Bradykinin (BK) and bradykinin-related peptides (BRPs), which were identified from a diversity of amphibian skin secretions, exerted contractile and relaxing effects on non-vascular and vascular smooth muscle, respectively. Here, we report a novel bradykinin-related peptide with a molecular mass of 1890.2 Da, RVAGPDKPARISGLSPLR, which was isolated and identified from Ordorrana hejiangensis skin secretions, followed by a C-terminal extension sequence VAPQIV. The biosynthetic precursor-encoding cDNA was cloned by the “shotgun” cloning method, and the novel RR-18 was identified and structurally confirmed by high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). Subsequently, the myotropic activity of the synthetic replicate of RR-18 was investigated on the rat bladder, uterus, tail artery and ileum smooth muscle. The peptide was named RR-18 in accordance (R = N-terminal arginine, R = C-terminal arginine, 18 = number of residues). In this study, the synthetic replicates of RR-18 showed no agonist/antagonism of BK-induced rat bladder and uterus smooth muscle contraction. However, it displayed an antagonism of bradykinin-induced rat ileum contraction and arterial smooth muscle relaxation. The EC50 values of BK for ileum and artery, were 214.7 nM and 18.3 nM, respectively. When the tissue was pretreated with the novel peptide, RR-18, at the maximally effective concentration of bradykinin (1 × 10−6 M), bradykinin-induced contraction of the ileum and relaxation of the arterial smooth muscle was reduced by 50–60% and 30–40%, respectively. In conclusion, RR-18 represents novel bradykinin antagonising peptide from amphibian skin secretions. It may provide new insight into possible treatment options for chronic pain and chronic inflammation.

Identification and pharmaceutical evaluation of novel frog skin-derived serine proteinase inhibitor peptide\u2013PE-BBI (Pelophylax esculentus Bowman-Birk inhibitor) for the potential treatment of cancer

Amphibian venom-derived peptides have high potential in the field of anticancer drug discovery. We have isolated a novel Bowman-Birk proteinase inhibitor (BBI)-type peptide from the skin secretion of Pelophylax esculentus (PE) named PE-BBI, and evaluated its bio-functions and anti-cancer activity in vitro. PE-BBI is a heptadecapeptide with C-terminal amidation. The mRNA sequence and primary structure of PE-BBI were identified using RT-PCR and LC/MS, respectively. A trypsin inhibitory assay was used to characterize the serine proteinase inhibitory activity of synthetic PE-BBI. PE-BBI’s myotropic activity was analyzed using isolated rat bladder and rat-tail artery smooth muscle tissues, and the anti-cancer ability of PE-BBI using human colorectal cancer cells. PE-BBI’s mechanism of action was investigated using Discovery studio software. PE-BBI showed trypsin inhibitory activity (Ki = 310 ± 72 nM), strong myotropic activity, and cytotoxicity that were specific to cancer cells, and no side effect to normal epithelial cells. The docking stimulation showed that PE-BBI had high affinity to several members of human kallikrein related peptidase (KLK) family. This finding helps to enrich our understanding of BBI peptides’ mode of action. Moreover, the data presented here validates frog secretions as sources of potential novel proteinase inhibitors for cancer treatment.

Identification of a Novel Vasodilatory Octapeptide from the Skin Secretion of the African Hyperoliid Frog, Kassina senegalensis

The defensive skin secretions of amphibians continue to be an excellent source of novel biologically-active peptides. Here we report the identification and pharmacological activity of a novel C-terminally amided myotropic octapeptide from the skin secretion of the African hyperoliid frog, Kassina senegalensis. The 8-amino acid peptide has the following primary structure: WMSLGWSL-amide and has a molecular mass of 978 Da. The primary structure and organisation of the biosynthetic precursor of WL-8 amide was successfully deduced from cloned skin secretion-derived cDNA. The open-reading frame encoded a single copy of WL-8, located at the C-terminus. Synthetic WL-8 amide was found to cause relaxation of rat tail artery smooth muscle with an EC50 of 25.98 nM. This peptide is unique in terms of its primary structure and is unlike any other peptide previously isolated from an amphibian source which has been archived in the NCBI database. WL-8 amide thus represents the prototype of a novel family of myotropic peptide from amphibian defensive skin secretions.

Ex Vivo Smooth Muscle Pharmacological Effects of a Novel Bradykinin-Related Peptide, and Its Analogue, from Chinese Large Odorous Frog, Odorrana livida Skin Secretions.

Bradykinin-related peptides (BRPs) are one of the most extensively studied frog secretions-derived peptide families identified from many amphibian species. The diverse primary structures of BRPs have been proven essential for providing valuable information in understanding basic mechanisms associated with drug modification. Here, we isolated, identified and characterized a dodeca-BRP (RAP-L1, T6-BK), with primary structure RAPLPPGFTPFR, from the skin secretions of Chinese large odorous frogs, Odorrana livida. This novel peptide exhibited a dose-dependent contractile property on rat bladder and rat ileum, and increased the contraction frequency on rat uterus ex vivo smooth muscle preparations; it also showed vasorelaxant activity on rat tail artery smooth muscle. In addition, the analogue RAP-L1, T6, L8-BK completely abolished these effects on selected rat smooth muscle tissues, whilst it showed inhibition effect on bradykinin-induced rat tail artery relaxation. By using canonical antagonist for bradykinin B1 or B2 type receptors, we found that RAP-L1, T6-BK -induced relaxation of the arterial smooth muscle was very likely to be modulated by B2 receptors. The analogue RAP-L1, T6, L8-BK further enhanced the bradykinin inhibitory activity only under the condition of co-administration with HOE140 on rat tail artery, suggesting a synergistic inhibition mechanism by which targeting B2 type receptors.

Anti-angiogenic activities of CRBGP from buccal glands of lampreys (Lampetra japonica).

Cysteine-rich secretory proteins (CRISPs), characterized by 16 conserved cysteines, are distributed in a wide range of organisms, such as secernenteas, amphibians, reptiles and mammals. In the previous studies, a novel CRISP family member (cysteine-rich buccal gland protein, CRBGP) was separated from the buccal gland of lampreys (Lampetra japonica, L.japonica). Lamprey CRBGP could not only suppress depolarization-induced contraction of rat tail arterial smooth muscle, but also block voltage-gated sodium channels (VGSCs). In the present study, the anti-angiogenic activities of lamprey CRBGP were investigated using endothelial cells and chick chorioallantoic membrane (CAM) models. Invitro assays, lamprey CRBGP is able to induce human umbilical vein endothelial cells (HUVECs) apoptosis by disturbing the calcium homeostasis and mitochondria functions. In addition, lamprey CRBGP could inhibit proliferation, adhesion, migration, invasion and tube formation of HUVECs by affecting the organization of F-actin and expression level of matrix metallo-proteinase 2 (MMP-2), matrix metallo-proteinase 9 (MMP-9) and vascular endothelial growth factor A (VEGFA) which are related to angiogenesis. Invivo assays, lamprey CRBGP could suppress the blood vessel formation in CAM models. Therefore, lamprey CRBGP is an important protein present in the buccal gland of lampreys and might help lampreys suppress the contraction of blood vessels, nociceptive responses and wound healing of host fishes during their feeding time. In addition, lamprey CRBGP might have the potential to act as an effective anti-angiogenic factor for the treatment of abnormal angiogenesis induced diseases.

Identification and functional analysis of a novel tryptophyllin peptide from the skin of the red-eye leaf frog, Agalychnis callidryas.

Amphibian skin has proved repeatedly to be a largely untapped source of bioactive peptides and this is especially true of members of the Phyllomedusinae subfamily of frogs native to South and Central America. Tryptophyllins are a group of peptides mainly found in the skin of members of this genus. In this study, a novel tryptophyllin (TPH) type 3 peptide, named AcT-3, has been isolated and structurally-characterised from the skin secretion and lyophilised skin extract of the red-eye leaf frog, Agalychnis callidryas. The peptide was identified in and purified from the skin secretion by reverse-phase HPLC. MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing established its primary structure as: pGlu-Gly-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Phe-Leu-Pro-Glu, with a non-protonated molecular mass of 1538.19Da. The mature peptide possessed the canonical N-terminal pGlu residue that arises from post-translational modification of a Gln residue. The deduced open-reading frame consisted of 63 amino acid residues encoding a highly-conserved signal peptide of approximately 22 amino acid residues, an intervening acidic spacer peptide domain, a single AcT-3 encoding domain and a C terminal processing site. A synthetic replicate of AcT-3 was found to antagonise the effect of BK on rat tail artery smooth muscle and to contract the intestinal smooth muscle preparations. It was also found that AcT-3 could dose-dependently inhibit the proliferation of human prostate cancer cell lines after 72h incubation.

A novel bradykinin-related dodecapeptide (RVALPPGFTPLR) from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis) exhibiting unusual structural and functional features.

Bradykinin-related peptides (BRPs) are significant components of the defensive skin secretions of many anuran amphibians, and these secretions represent the source of the most diverse spectrum of such peptides so far encountered in nature. Of the many families of bioactive peptides that have been identified from this source, the BRPs uniquely appear to represent homologues of counterparts that have specific distributions and receptor targets within discrete vertebrate taxa, ranging from fishes through mammals. Their broad spectra of actions, including pain and inflammation induction and smooth muscle effects, make these peptides ideal weapons in predator deterrence. Here, we describe a novel 12-mer BRP (RVALPPGFTPLR-RVAL-(L1, T6, L8)-bradykinin) from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis). The C-terminal 9 residues of this BRP (-LPPGFTPLR) exhibit three amino acid substitutions (L/R at Position 1, T/S at Position 6 and L/F at Position 8) when compared to canonical mammalian bradykinin (BK), but are identical to the kinin sequence present within the cloned kininogen-2 from the Chinese soft-shelled turtle (Pelodiscus sinensis) and differ from that encoded by kininogen-2 of the Tibetan ground tit (Pseudopodoces humilis) at just a single site (F/L at Position 8). These data would imply that the novel BRP is an amphibian defensive agent against predation by sympatric turtles and also that the primary structure of the avian BK, ornithokinin (RPPGFTPLR), is not invariant within this taxon. Synthetic RVAL-(L1, T6, L8)-bradykinin was found to be an antagonist of BK-induced rat tail artery smooth muscle relaxation acting via the B2-receptor.

Ranakinestatin-PPF from the skin secretion of the Fukien gold-striped pond frog, Pelophylax plancyi fukienensis: a prototype of a novel class of bradykinin B2 receptor antagonist peptide from ranid frogs.

The defensive skin secretions of many amphibians are a rich source of bradykinins and bradykinin-related peptides (BRPs). Members of this peptide group are also common components of reptile and arthropod venoms due to their multiple biological functions that include induction of pain, effects on many smooth muscle types, and lowering systemic blood pressure. While most BRPs are bradykinin receptor agonists, some have curiously been found to be exquisite antagonists, such as the maximakinin gene-related peptide, kinestatin—a specific bradykinin B2-receptor antagonist from the skin of the giant fire-bellied toad, Bombina maxima. Here, we describe the identification, structural and functional characterization of a heptadecapeptide (DYTIRTRLHQGLSRKIV), named ranakinestatin-PPF, from the skin of the Chinese ranid frog, Pelophylax plancyi fukienensis, representing a prototype of a novel class of bradykinin B2-receptor specific antagonist. Using a preconstricted preparation of rat tail arterial smooth muscle, a single dose of 10−6 M of the peptide effectively inhibited the dose-dependent relaxation effect of bradykinin between 10−11 M and 10−5 M and subsequently, this effect was pharmacologically-characterized using specific bradykinin B1- (desArg-HOE140) and B2-receptor (HOE140) antagonists; the data from which demonstrated that the antagonism of the novel peptide was mediated through B2-receptors. Ranakinestatin—PPF—thus represents a prototype of an amphibian skin peptide family that functions as a bradykinin B2-receptor antagonist herein demonstrated using mammalian vascular smooth muscle.

PsT-1: A new tryptophyllin peptide from the skin secretion of Waxy Monkey Leaf Frog, Phyllomedusa sauvagei

The Waxy Monkey Leaf Frog, Phyllomedusa sauvagei, has been extensively-studied for many years, and a broad spectrum of bioactive peptides has been found in its skin secretions. Here we report the discovery of a novel tryptophyllin (TPH) peptide, named PsT-1, from this frog species. Skin secretions from specimens of P. sauvagei were collected by mild electrical stimulation. Peptides were identified and characterized by transcriptome cloning, and the structure was confirmed by MALDI-TOF mass spectrometry and automated Edman degradation. This novel peptide was encoded by a single precursor of 61 amino acid residues, whose primary structure was deduced from cloned skin cDNA. Analysis of different amphibian tryptophyllins revealed that PsT-1 exhibited a high degree of primary structural similarity to its homologs, PdT-1 and PdT-2, from the Mexican giant leaf frog, Pachymedusa dacnicolor. A synthetic replicate of PsT-1 was found to inhibit bradykinin-induced vasorelaxation of phenylephrine pre-constricted rat tail artery smooth muscle. It was also found that PsT-1 had an anti-proliferative effect on three different human prostate cancer cell lines (LNCaP/PC3/DU145), by use of an MTT assay coupled with direct cell counting as measures of cell growth. These data indicate that PsT-1 is a likely bradykinin receptor antagonist and its biological effects are probably mediated through bradykinin receptors. As a BK antagonist, PST-1, with antagonistic effects on BK in artery smooth muscle, inhibition of proliferation in prostate cancer cells and lack of undesirable side effects, may have potential in cardiovascular, inflammatory and anticancer therapy.

Senegalin: a novel antimicrobial/myotropic hexadecapeptide from the skin secretion of the African running frog, Kassina senegalensis

Amphibian skin is a rich and unique source of novel bioactive peptides most of which are endowed with either antimicrobial or pharmacological properties. Here, we report the identification and structural characterization of a novel peptide, named senegalin, which possesses both activities. Senegalin is a hexadecapeptide amide (FLPFLIPALTSLISSL-NH2) of unique primary structure found in the skin secretion of the African running frog, Kassina senegalensis. The structure of the biosynthetic precursor of senegalin, deduced from cloned skin cDNA, consists of 76 amino acid residues and displays the typical domain organization of an amphibian skin peptide precursor. Both natural senegalin and its synthetic replicate displayed antimicrobial and myotropic activities. Senegalin was active against Staphylococcus aureus (MIC 50μM) and Candida albicans (MIC 150μM) but was non-haemolytic at concentrations up to and including 150μM. In contrast, senegalin induced a dose-dependent contraction of rat urinary bladder smooth muscle (EC50 2.9nM) and a dose-dependent relaxation of rat tail artery smooth muscle (EC50 37.7nM). Senegalin thus represents a prototype biologically active amphibian skin peptide and illustrates the fact that amphibian skin secretion peptidomes continue to be unique sources of such molecules.

High-Dose, but Not Low-Dose, Aspirin Impairs Anticontractile Effect of Ticagrelor following ADP Stimulation in Rat Tail Artery Smooth Muscle Cells

Objective. To compare effects of low- versus high-dose aspirin coadministered with ticagrelor on the reactivity of vascular smooth muscle cells (VSMCs). Methods. Wistar rats were orally administered ticagrelor (10 mg/kg) and/or aspirin (2 or 10 mg/kg) (n = 7 per each of 4 groups) or placebo (n = 9) 12 and 2 hours before experiments. Anticontractile effects of ticagrelor were assessed in perfusion solution containing ticagrelor (1 μM/L). Changes in perfusion pressure proportional to the degree of adenosine diphosphate analogue- (2-MeS-ADP-) and phenylephrine-induced constriction of rat tail arteries were evaluated. Results. Pretreatment with high- but not low-dose aspirin enhanced the reactivity of VSMCs only in endothelium-lined vessels. Suppression of 2-MeS-ADP-induced VSMC contraction by ticagrelor observed in arteries with and without endothelium was maintained in endothelialized arteries pretreated only with low-dose aspirin. For endothelium-denuded vessels and low-dose aspirin we observed a significant reduction of the maximal effect of ticagrelor with no rightward shift of the concentration-response curve for phenylephrine. With high-dose aspirin pretreatment ticagrelor exerted no anticontractile effect. Conclusion. High-dose, but not low-dose, aspirin impairs the anticontractile effect of ticagrelor on ADP-induced VSMC contraction in the rat model. Both the clinical significance and detailed underlying mechanism of our findings require further investigation.

The natriuretic peptide/helokinestatin precursor from Mexican beaded lizard ( Heloderma horridum) venom: Amino acid sequence deduced from cloned cDNA and identification of two novel encoded helokinestatins

Natriuretic peptides are common components of reptile venoms and molecular cloning of their biosynthetic precursors has revealed that in snakes, they co-encode bradykinin-potentiating peptides and in venomous lizards, some co-encode bradykinin inhibitory peptides such as the helokinestatins. The common natriuretic peptide/helokinestatin precursor of the Gila Monster, Heloderma suspectum, encodes five helokinestatins of differing primary structures. Here we report the molecular cloning of a natriuretic peptide/helokinestatin precursor cDNA from a venom-derived cDNA library of the Mexican beaded lizard ( Heloderma horridum). Deduction of the primary structure of the encoded precursor protein from this cloned cDNA template revealed that it consisted of 196 amino acid residues encoding a single natriuretic peptide and five helokinestatins. While the natriuretic peptide was of identical primary structure to its Gila Monster ( H. suspectum) homolog, the encoded helokinestatins were not, with this region of the common precursor displaying some significant differences to its H. suspectum homolog. The helokinestatin-encoding region contained a single copy of helokinestatin-1, 2 copies of helokinestatin-3 and single copies of 2 novel peptides, (Phe) 5-helokinestatin-2 (VPPAFVPLVPR) and helokinestatin-6 (GPPFNPPPFVDYEPR). All predicted peptides were found in reverse phase HPLC fractions of the same venom. Synthetic replicates of both novel helokinestatins were found to antagonize the relaxing effect of bradykinin on rat tail artery smooth muscle. Thus lizard venom continues to provide a source of novel biologically active peptides.

Peptide IC-20, encoded by skin kininogen-1 of the European yellow-bellied toad, Bombina variegata, antagonizes bradykinin-induced arterial smooth muscle relaxation

Objectives:The objectives were to determine if the skin secretion of the European yellow-bellied toad (Bombina variegata), in common with other related species, contains a bradykinin inhibitor peptide and to isolate and structurally characterize this peptide.Materials and Methods:Lyophilized skin secretion obtained from this toad was subjected to reverse phase HPLC fractionation with subsequent bioassay of fractions for antagonism of the bradykinin activity using an isolated rat tail artery smooth muscle preparation. Subsequently, the primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy, and molecular cloning, following which a synthetic replicate was chemically synthesised for bioassay.Results:A single peptide of molecular mass 2300.92 Da was resolved in HPLC fractions of skin secretion and its primary structure determined as IYNAIWP-KH-NK-KPGLL-. Database interrogation with this sequence indicated that this peptide was encoded by skin kininogen-1 previously cloned from B. variegata. The blank cycles were occupied by cysteinyl (C) residues and the peptide was located toward the C-terminus of the skin kininogen, and flanked N-terminally by a classical –KR- propeptide convertase processing site. The peptide was named IC-20 in accordance (I = N-terminal isoleucine, C = C-terminal cysteine, 20 = number of residues). Like the natural peptide, its synthetic replicate displayed an antagonism of bradykinin-induced arterial smooth muscle relaxation.Conclusion:IC-20 represents a novel bradykinin antagonizing peptide from amphibian skin secretions and is the third such peptide found to be co-encoded with bradykinins within skin kininogens.

The structure of helokinestatin-5 and its biosynthetic precursor from Gila monster ( Heloderma suspectum) venom: Evidence for helokinestatin antagonism of bradykinin-induced relaxation of rat tail artery smooth muscle

Here we report the primary structure of a novel peptide, named helokinestatin-5 (VPPPLQMPLIPR), from the venom of the Gila monster ( Heloderma suspectum). Helokinestatin-5 differs in structure from helokinestatin-3 by deletion of a single prolyl residue in the N-terminally located polyproline region. Two different biosynthetic precursors were consistently cloned from a venom-derived cDNA library. The first encoded helokinestatins 1–4 and a single copy of C-type natriuretic peptide, as previously described, whereas the second was virtually identical, lacking only a single prolyl codon as found in the mature attenuated helokinestatin-5 peptide. Helokinestatins 1–3 and 5 were synthesized by solid-phase fmoc chemistry and each synthetic replicate was found to antagonize the relaxation effect induced by bradykinin on rat tail artery smooth muscle. Helokinestatins thus represent a novel family of vasoactive peptides from the venom of helodermatid lizards.

Peptide DV-28 amide: An inhibitor of bradykinin-induced arterial smooth muscle relaxation encoded by Bombina orientalis skin kininogen-2

Skin kininogens from bombinid toads encode an array of bradykinin-related peptides and one such kininogen from Bombina maxima also encodes the potent bradykinin B2-receptor antagonist, kinestatin. In order to determine if the skin secretion of the closely-related toad, Bombina orientalis, contained a bradykinin inhibitory peptide related to kinestatin, we screened reverse phase HPLC fractions of defensive skin secretion using a rat tail artery smooth muscle preparation. A fraction was located that inhibited bradykinin-induced relaxation of the preparation and this contained a peptide of 3198.5 Da as determined by MALDI-TOF MS. Automated Edman degradation of this peptide established the identity of a 28-mer as: DMYEIKGFKSAHGRPRVCPPGEQCPIWV, with a disulfide-bridge between Cys 18 and Cys 24 and an amidated C-terminal Val residue. Peptide DV-28 was found to correspond to residues 133–160 of skin pre-kininogen-2 of B. orientalis that also encodes two copies of (Thr 6)-bradykinin. The C-terminal residue, Gly-161, of the precursor open-reading frame, acts as the C-terminal amide donor of mature DV-28. DV-28 amide thus represents a new class of bradykinin inhibitor peptide from amphibian skin secretion.

Helokinestatin: A new bradykinin B 2 receptor antagonist decapeptide from lizard venom

Synthetic bradykinin antagonist peptides/peptoids have been powerful tools for delineating the roles of kinins in both normal physiology and in pathological states. Here, we report the identification of a novel, naturally occurring bradykinin B 2 receptor antagonist peptide, helokinestatin, isolated and structurally characterized from the venoms of helodermatid lizards—the Gila monster ( Heloderma suspectum) and the Mexican beaded lizard ( Heloderma horridum). The primary structure of the peptide was established by a combination of microsequencing and mass spectroscopy as Gly-Pro-Pro-Tyr-Gln-Pro-Leu-Val-Pro-Arg ( M r 1122.62). A synthetic replicate of helokinestatin was found to inhibit bradykinin-induced vasorelaxation of phenylephrine pre-constricted rat tail artery smooth muscle, mediated by the B 2 receptor sub-type, in a dose-dependent manner. Natural selection, that generates functional optimization of predatory reptile venom peptides, can potentially provide new insights for drug lead design or for normal physiological or pathophysiological processes.

Novel cysteine-rich secretory protein in the buccal gland secretion of the parasitic lamprey, Lethenteron japonicum

Lampreys are one of the most primitive vertebrates diverged some 500 million years ago. It has long been known that parasitic lampreys secrete anticoagulant from their buccal glands and prevent blood coagulation of host fishes. We found two major protein components of 160 and 26 kDa in the buccal gland secretion of parasitic river lamprey, Lethenteron japonicum. The larger protein was identified as river lamprey plasma albumin. The complete primary structure of the 26-kDa protein was determined by protein and cDNA analysis. It belonged to the cysteine-rich secretory protein (CRISP) superfamily that includes recently identified reptile venom ion-channel blockers. Lamprey CRISP blocked depolarization-induced contraction of rat-tail arterial smooth muscle, but showed no effect on caffeine-induced contraction. The result suggests that lamprey CRISP is an L-type Ca 2+-channel blocker and may act as a vasodilator, which facilitates the parasite to feed on the host’s blood. The lamprey CRISP protein contains a number of short insertions throughout the sequence, when aligned with reptilian venom CRISP proteins, probably due to the large evolutionary distance between the Agnatha and the Reptilia, and may represent a novel class of venom CRISP family proteins.

Rho-kinase-mediated regulation of receptor-agonist-stimulated smooth muscle contraction

Rho kinase was shown to regulate smooth muscle contraction through modulating myosin phosphatase (MLCP) activity, but the in vivo mechanism remains to be clarified. This study examined the effects of Rho kinase inhibition on the phosphorylation time course of MLCP subunit MYPT1 at Thr697 and Thr855 and MLCP inhibitory protein CPI-17 at Thr38 and on actin polymerization during the contraction of rat tail artery (RTA) smooth muscle. Rho kinase inhibitor Y27632 suppressed force activated by alpha(1)-adrenergic agonist phenylephrine or thromboxane A(2) analog U46619 with concomitant decreases in MLC(20) phosphorylation. Phenylephrine and U46619 significantly increased MYPT1(Thr855) phosphorylation that was eliminated by Y27632 pretreatment, whereas MYPT1(Thr697) phosphorylation was not stimulated. Phenylephrine increased CPI-17(Thr38) phosphorylation that was not inhibited by Y27632 but was abolished by a protein kinase C inhibitor Ro 31-8220; in contrast, U46619 did not stimulate CPI-17 phosphorylation. Both agonists increased actin polymerization that was diminished by Y27632 under phenylephrine but not U46619 activation. These results demonstrated a temporal correlation between MYPT1(Thr855) phosphorylation, MLC(20) phosphorylation, and contraction in a Rho-kinase-dependent manner for both phenylephrine and U46619 stimulation, suggesting that Rho kinase regulates MLCP activity through MYPT1(Thr855) phosphorylation during RTA smooth muscle contraction. Furthermore, Rho kinase regulates actin polymerization activated by alpha(1)-adrenoceptors but is less significant in thromboxane receptor stimulation.

Extracellular signal-regulated kinase1/2 in contraction of vascular smooth muscle

Smooth muscle contractility is regulated by both intracellular Ca 2+ concentration ([Ca 2+] i) and Ca 2+ sensitivity of the contractile apparatus. Extracellular signal-regulated kinases1/2 (ERK1/2) have been implicated in modulating Ca 2+ sensitivity of smooth muscle contraction but mechanisms of action remain elusive. This study investigated the roles of ERK1/2 in modulating [Ca 2+] i, calcium sensitivity and the 20-kDa myosin light chain (MLC 20) phosphorylation during contraction activated by α 1-adrenoceptor agonist phenylephrine and thromboxane A 2 mimetic U46619 in rat tail artery strips. A specific inhibitor for ERK1/2 activation, U0126, inhibited phenylephrine- and U46619-induced contraction, shifting both concentration-response curves rightward. During phenylephrine-stimulated contraction, U0126 exhibited concentration-dependent inhibition towards force but significant decreases in [Ca 2+] i were detected only at higher concentration. Both phenylephrine and U46619 induced a transient activation of ERK1/2 which was abolished by U0126 but unaffected by a general tyrosine kinase inhibitor genistein or Rho kinase inhibitor Y27632 at concentrations inhibiting more than 50% force. Interestingly, U0126 had no effect on steady-state MLC 20 phosphorylation levels stimulated by both receptor agonists. These results indicated that during contraction of rat tail artery smooth muscle activated by α 1-adrenoceptor agonist or thromboxane A 2 analogue, ERK1/2 increase Ca 2+ sensitivity that does not involve the modulation of MLC 20 phosphorylation.