Panot Tangsucharit

Hyperinsulinemia induces hypertension associated with neurogenic vascular dysfunction resulting from abnormal perivascular innervations in rat mesenteric resistance arteries

We previously reported that chronic hyperinsulinemia and insulin resistance induced by fructose-drinking loading elicited hypertension associated with abnormal neuronal regulation of vascular tone in an in vivo study using pithed rats. Therefore, to further clarify the detailed mechanisms of perivascular nervous system malfunction induced by chronic hyperinsulinemia, we investigated the neurogenic vascular responses and distribution of perivascular nerves using mesenteric vascular beds isolated from fructose-loaded rats with hyperinsulinemia. Male Wistar rats (6 weeks old) received 15% fructose solution as drinking fluid for 10 weeks (fructose-drinking rats, FDR), which resulted in significant increases in plasma levels of insulin, the glucose-insulin index, blood norepinephrine (NE) levels and systolic blood pressure, but not blood glucose levels, when compared with normal water-drinking rats (control rats). In perfused mesenteric vascular beds of FDR, enhanced adrenergic nerve-mediated vasoconstriction with no effect on NE-induced vasoconstriction and decreased calcitonin gene-related peptide (CGRP)-containing nerve-mediated vasodilation with no effect on CGRP-induced vasodilation were observed. Immunohistochemistry studies showed increased density of neuropeptide Y immunopositive adrenergic fibers and reduced density of CGRP immunopositive fibers in mesenteric arteries of FDR. Furthermore, FDR showed decreased CGRP content in dorsal root ganglia. These findings suggest that dysfunction of the neuronal vascular control system resulting from abnormal innervation of mesenteric perivascular nerves induced by the hyperinsulinemic state is responsible for the development of hypertension in FDR.

Paracrine control of mesenteric perivascular axo-axonal interaction

Immunohistochemical study of rat mesenteric arteries showed dense innervation of adrenergic nerves, calcitonin gene‐related peptide (CGRP)‐containing nerves (CGRPergic nerves), nitric oxide‐containing nerves (nitrergic nerves). Double‐immunostaining revealed that most CGRPergic or nitrergic nerves were in close contact with adrenergic nerves. CGRPergic and transient receptor potential vanilloid‐1 (TRPV1)‐immunopositive nerves appeared in the same neurone. In rat perfused mesenteric vascular beds without endothelium and with active tone, perfusion of nicotine, or bolus injection of capsaicin and acetylcholine and periarterial nerve stimulation (PNS) lowered pH levels of out flowed perfusate concomitant with vasodilation. Cold‐storage denervation of preparations abolished pH lowering induced by nicotine and PNS. Guanethidine inhibited PNS‐ and nicotine‐, but not acetylcholine‐ and capsaicin‐, induced pH lowering. Pharmacological analysis showed that protons were released not only from adrenergic nerves but also from CGRPergic nerves. A study using a fluorescent pH indicator demonstrated that nicotine, acetylcholine and capsaicin applied outside small mesenteric artery lowered perivascular pH levels, which were not observed in Ca2+ free medium. Exogenously injected hydrochloric acid in denuded preparations induced pH lowering and vasodilation, which was inhibited by denervation, TRPV1 antagonists and capsaicin without affecting pH lowering. These results suggest that excitement of adrenergic nerves releases protons to activate TRPV1 in CGRPergic nerves and thereby induce vasodilation. It is also suggested that CGRPergic nerves release protons with exocytosis to facilitate neurotransmission via a positive feedback mechanism.

Histamine H3 receptor-mediated modulation of perivascular nerve transmission in rat mesenteric arteries

The rat mesenteric artery has been shown to be innervated by adrenergic vasoconstrictor nerves and calcitonin gene-related peptide (CGRP)-containing (CGRPergic) vasodilator nerves. The present study was designed to investigate the involvement of histamine H(3) receptors in the neurotransmission of perivascular adrenergic and CGRPergic nerves. The mesenteric vascular beds without an endothelium isolated from male Wistar rats were perfused with Krebs solution and perfusion pressure was measured. In preparations with resting tension, the selective H(3) receptor agonist (R)-α-methylhistamine (α-methylhistamine; 10-100nM) significantly reduced periarterial nerve stimulation (2-8Hz)-induced vasoconstriction and noradrenaline release in the perfusate without an effect on the vasoconstriction induced by exogenously injected noradrenaline (0.5, 1.0nmol). In preparations with active tone produced by methoxamine (2μM) and in the presence of guanethidine (5μM), the periarterial nerve stimulation (1, 2Hz)-induced vasodilator response was inhibited by α-methylhistamine (0.1-1μM) perfusion without affecting vasodilation induced by exogenously injected CGRP (5pmol). Clobenpropit (histamine H(3) receptor antagonist, 1μM) canceled the α-methylhistamine-induced decrease in the periarterial nerve stimulation-induced vasoconstriction and noradrenaline release and periarterial nerve stimulation-induced vasodilation. These results suggest that the stimulation of H(3) receptors located in rat perivascular nerves inhibits presynaptically the neurotransmission of not only adrenergic nerves, but also CGRP nerves, by decreasing neurotransmitters.

Synergistic Antihypertensive Effect of Carthamus tinctorius L. Extract and Captopril in l-NAME-Induced Hypertensive Rats via Restoration of eNOS and AT1R Expression

This study examined the effect of Carthamus tinctorius (CT) extract plus captopril treatment on blood pressure, vascular function, nitric oxide (NO) bioavailability, oxidative stress and renin-angiotensin system (RAS) in Nω-Nitro-l-arginine methyl ester (l-NAME)-induced hypertension. Rats were treated with l-NAME (40 mg/kg/day) for five weeks and given CT extract (75 or 150 or 300 or 500 mg/kg/day): captopril (5 mg/kg/day) or CT extract (300 mg/kg/day) plus captopril (5 mg/kg/day) for two consecutive weeks. CT extract reduced blood pressure dose-dependently, and the most effective dose was 300 mg/kg/day. l-NAME-induced hypertensive rats showed abnormalities including high blood pressure, high vascular resistance, impairment of acetylcholine-induced vasorelaxation in isolated aortic rings and mesenteric vascular beds, increased vascular superoxide production and plasma malondialdehyde levels, downregulation of eNOS, low level of plasma nitric oxide metabolites, upregulation of angiotensin II type 1 receptor and increased plasma angiotensin II. These abnormalities were alleviated by treatment with either CT extract or captopril. Combination treatment of CT extract and captopril normalized all the abnormalities found in hypertensive rats except endothelial dysfunction. These data indicate that there are synergistic antihypertensive effects of CT extract and captopril. These effects are likely mediated by their anti-oxidative properties and their inhibition of RAS.

Involvement of perivascular nerves and transient receptor potential vanilloid 1 (TRPV1) in vascular responses to histamine in rat mesenteric resistance arteries

A previous report showed that histamine in denuded mesenteric vascular beds produced a triphasic response; an initial small histamine H(2) receptor-mediated vasodilation, a transient histamine H(1) receptor-mediated vasoconstriction, and finally a long-lasting vasodilation. We further investigated the vascular effect of histamine in mesenteric preparations without an endothelium to clarify the possible involvement of perivascular nerves. Male Wistar rat mesenteric vascular beds without an endothelium were perfused with Krebs solution containing methoxamine to produce active tone and lafutidine to block histamine H(2) receptor-mediated vasodilation. Histamine (1-100μM) was perfused for 1min and perfusion pressure was measured with a pressure transducer. Histamine caused a biphasic vascular response; initial vasoconstriction followed vasodilation. Tetrodotoxin (a neurotoxin, 1μM) and procaine (a local anesthetic, 100μM) significantly inhibited the vasoconstriction and vasodilation. Ruthenium red (a transient receptor potential vanilloid 1 (TRPV1) antagonist, 1μM) also significantly inhibited both phases of the response. Pretreatment with capsaicin (a depletor of calcitonin gene-related peptide (CGRP)-containing nerves, 5μM) significantly inhibited the vasodilation without affecting the initial vasoconstriction. Both indomethacin (a cyclooxygenase inhibitor, 0.5μM) and seratrodast (a thromboxane A(2) receptor antagonist, 0.1μM) abolished the histamine-induced vasoconstriction and subsequent vasodilation. These results suggest that histamine-induced vasoconstriction and long-lasting vasodilation are mediated by activation of TRPV1 on capsaicin-sensitive and capsaicin-insensitive nerves. They also suggest that perivascular nerves and prostanoids, probably thromboxane A(2), are responsible for the vascular response to histamine.

Adrenergic stimulation-released 5-HT stored in adrenergic nerves inhibits CGRPergic nerve-mediated vasodilatation in rat mesenteric resistance arteries.

5‐HT is taken up by and stored in adrenergic nerves and periarterial nerve stimulation (PNS) releases 5‐HT to cause vasoconstriction in rat mesenteric arteries. The present study investigated whether PNS‐released 5‐HT stored in adrenergic nerves affects the function of perivascular calcitonin gene‐related peptide‐containing (CGRPergic) nerves.

Muscarinic acetylcholine receptor M1 and M3 subtypes mediate acetylcholine-induced endothelium-independent vasodilatation in rat mesenteric arteries

The present study investigated pharmacological characterizations of muscarinic acetylcholine receptor (AChR) subtypes involving ACh-induced endothelium-independent vasodilatation in rat mesenteric arteries. Changes in perfusion pressure to periarterial nerve stimulation and ACh were measured before and after the perfusion of Krebs solution containing muscarinic receptor antagonists. Distributions of muscarinic AChR subtypes in mesenteric arteries with an intact endothelium were studied using Western blotting. The expression level of M1 and M3 was significantly greater than that of M2. Endothelium removal significantly decreased expression levels of M2 and M3, but not M1. In perfused mesenteric vascular beds with intact endothelium and active tone, exogenous ACh (1, 10, and 100 nmol) produced concentration-dependent and long-lasting vasodilatations. In endothelium-denuded preparations, relaxation to ACh (1 nmol) disappeared, but ACh at 10 and 100 nmol caused long-lasting vasodilatations, which were markedly blocked by the treatment of pirenzepine (M1 antagonist) or 4-DAMP (M1 and M3 antagonist) plus hexamethonium (nicotinic AChR antagonist), but not methoctramine (M2 and M4 antagonist). These results suggest that muscarinic AChR subtypes, mainly M1, distribute throughout the rat mesenteric arteries, and that activation of M1 and/or M3 which may be located on CGRPergic nerves releases CGRP, causing an endothelium-independent vasodilatation.

Do cholinergic nerves innervating rat mesenteric arteries regulate vascular tone

Vascular blood vessels have various types of cholinergic acetylcholine receptors (AChR), but the source of ACh has not been confirmed. Perivascular adrenergic nerves and nonadrenergic calcitonin gene-related peptide (CGRP)-containing (CGRPergic) nerves innervate rat mesenteric arteries and regulate vascular tone. However, function of cholinergic innervation remains unknown. The present study investigated cholinergic innervation by examining effects of cholinesterase inhibitor (neostigmine), a muscarinic AChR antagonist (atropine), and a nicotinic AChR antagonist (hexamethonium) on adrenergic nerve-mediated vasoconstriction and CGRPergic nerve-mediated vasodilation in rat mesenteric vascular beds without endothelium. In preparations treated with capsaicin (CGRP depletor) or in the presence of N(ω)-nitro-l-arginine methyl ester (nonselective nitric oxide synthase inhibitor), perivascular nerve stimulation (PNS; 2-12 Hz) evoked a frequency-dependent vasoconstriction. In the same preparations, exogenous norepinephrine induced a concentration-dependent vasoconstriction. Atropine, hexamethonium, and neostigmine had no effect on vasoconstrictor responses to PNS and norepinephrine injections. In denuded preparations, these cholinergic agents did not affect the PNS (12 Hz)-evoked release of norepinephrine in perfusate. In preconstricted preparations without endothelium in the presence of guanethidine (adrenergic neuron blocker), PNS (1-4 Hz) induced a frequency-dependent vasodilation, which was not affected by atropine, hexamethonium, and neostigmine. In denuded preparations treated with capsaicin and guanethidine, PNS did not induce vascular responses, and atropine, neostigmine, and physostigmine had no effect on PNS. Immunohistochemistry study showed choline acetyltransferase-immunopositive fibers, which were resistant to capsaicin and 6-hydroxydopamine (adrenergic toxin). These results suggest that rat mesenteric arteries have cholinergic innervation, which is different from adrenergic and capsaicin-sensitive nerves and not associated with vascular tone regulation.

Protons modulate perivascular axo‐axonal neurotransmission in the rat mesenteric artery

Previous studies have demonstrated that nicotine releases protons from adrenergic nerves via stimulation of nicotinic ACh receptors and activates transient receptor potential vanilloid‐1 (TRPV1) receptors located on calcitonin gene‐related peptide (CGRP)‐containing (CGRPergic) vasodilator nerves, resulting in vasodilatation. The present study investigated whether perivascular nerves release protons, which modulate axon‐axonal neurotransmission.

Increase in L-type amino acid transporter 1 expression during cholangiocarcinogenesis caused by liver fluke infection and its prognostic significance

L-type amino acid transporter 1 (LAT1) is highly expressed in various human cancers, including cholangiocarcinoma (CCA), the most common cancer in Northeast Thailand. Chronic inflammation and oxidative stress induced by liver fluke, Opisthorchis viverrini, infection has been recognized as the major cause of CCA in this area. We show here that an increased expression of LAT1 and its co-functional protein CD98 are found during carcinogenesis induced by Ov in hamster CCA tissues. We also demonstrate that oxidative stress induced by H2O2 is time-dependent and dramatically activates LAT1 and CD98 expression in immortal cholangiocytes (MMNK1). In addition, H2O2 treatment increased LAT1 and CD98 expression, as well as an activated form of AKT and mTOR in MMNK1 and CCA cell lines (KKU-M055 and KKU-M213). We also show that suppression of PI3K/AKT pathway activity with a dual PI3K/mTOR inhibitor, BEZ235, causes a reduction in LAT1 and CD98 expression in KKU-M055 and KKU-M213 in parallel with a reduction of activated AKT and mTOR. Interestingly, high expression of LAT1 in human CCA tissues is a significant prognostic factor for shorter survival. Taken together, our data show that LAT1 expression is significantly associated with CCA progression and cholangiocarcinogenesis induced by oxidative stress. Moreover, the expression of LAT1 and CD98 in CCA is possibly regulated by the PI3K/AKT signaling pathway.