Koichiro Takasaki

Adrenergic modulation of calcitonin gene-related peptide (CGRP)-containing nerve-mediated vasodilation in the rat mesenteric resistance vessel

Pharmacological studies showed that periarterial nerve stimulation (PNS) of the perfused rat mesenteric vascular bed contracted with endothelin, a vasoconstrictor peptide, in the presence of prazosin (alpha 1-adrenoceptor antagonist) produced a frequency-dependent neurogenic vasodilation when the adrenergic neurotransmission was blocked by the adrenergic neuron blocker, guanethidine. The PNS-evoked vasodilation was attenuated by tetrodotoxin and capsaicin treatment, and was also inhibited when the adrenergic neurotransmitter (norepinephrine; NE) release was left intact in the absence of guanethidine. However, in the combined presence of an alpha 2-adrenoceptor antagonist (yohimbine) and prazosin, PNS caused a marked neurogenic vasodilation even when the neuronal release of NE was left intact. These results suggest that NE released from adrenergic nerves regulates the release of a vasodilator substance, CGRP, through activation of alpha 2-adrenoceptors on CGRP-containing vasodilator nerves.

Endogenous calcitonin gene-related peptide mediates nonadrenergic noncholinergic depressor response to spinal cord stimulation in the pithed rat.

The role of endogenous calcitonin gene-related peptide (CGRP) in the nonadrenergic noncholinergic depressor response to spinal cord stimulation was studied in the pithed rat in vivo. Pithed rats were given hexamethonium (2 mg/kg per minute i.v.) to block autonomic outflow, and mean blood pressure was artificially maintained at approximately 100 mm Hg with methoxamine (10-15 micrograms/kg per minute i.v.). Electrical stimulation of the spinal cord at the level of the lower thoracic vertebra (T9-12) caused a fall in blood pressure in a frequency-dependent (0.5-10 Hz), voltage-dependent (2.5-50 V), and pulse duration-dependent (0.25-8 msec) manner. The heart rate did not change during the depressor response. The depressor response was long lasting, and the maximum response was elicited by stimulation at 4-6 Hz. The neurotoxin tetrodotoxin (100 micrograms/kg i.v.) abolished the depressor response to spinal cord stimulation, whereas treatment with propranolol (0.5 mg/kg per minute i.v.), atropine (0.05 mg/kg per minute i.v.), or a combination of pyrilamine (0.5 mg/kg per minute i.v.) and cimetidine (0.5 mg/kg per minute i.v.) did not affect the response. In pithed rats treated with capsaicin (total dose of 500 mg/kg s.c.), spinal cord stimulation caused a slight depressor response. Exogenous CGRP, but not acetylcholine, isoproterenol, histamine, or substance P, caused a sustained fall in blood pressure that mimicked the spinal cord stimulation-induced depressor response. Continuous infusion of CGRP[8-37] (60 nmol/kg per minute i.v.), a CGRP receptor antagonist, markedly inhibited the depressor responses not only to spinal cord stimulation but also to exogenous CGRP.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of endothelium removal on the vasoconstrictor response to neuronally released 5‐hydroxytryptamine and noradrenaline in the rat isolated mesenteric and femoral arteries

1 The role of the vascular endothelium in the vasoconstrictor response to transmural nerve stimulation (TNS) was studied in isolated ring segments of rat mesenteric and femoral arteries. 2 In both types of artery, TNS (1 to 16 Hz) produced frequency‐dependent vasoconstriction, which was abolished by 100 nm tetrodotoxin, 10 μm guanethidine or 10 nm prazosin, indicating that the response was mediated by endogenous noradrenaline (NA) released from noradrenergic nerves. NA‐mediated vasoconstriction in response to TNS was significantly potentiated by removal of the endothelium. 3 In the presence of 10 nm prazosin, the reduced vasoconstriction in response to TNS was restored by incubation with 10 μm 5‐hydroxytryptamine (5‐HT) for 20 min. Restoration of the response to TNS was markedly attenuated by treatment with 10 nm ketanserin, 100 nm tetrodotoxin, or 10 μm guanethidine, indicating that the restored response was mediated by 5‐HT released from noradrenergic nerves. Vasoconstriction mediated by 5‐HT in response to TNS was not modified by removal of the endothelium. 4 In both types of artery with intact endothelium, treatment with 3μm methylene blue potentiated the NA‐mediated contractile response to TNS, but did not potentiate the 5‐HT‐mediated response to TNS. 5 In both types of artery, the contractile responses to exogenous NA and 5‐HT were potentiated by removal of the endothelium. 6 These results suggest that endothelial cells regulate neurogenic vasoconstriction by releasing endothelium‐derived relaxing factor. Furthermore, it appears likely that the response to neuronally released 5‐HT is not affected by the endothelium.

Changes in calcitonin gene-related peptide (CGRP)-containing vasodilator nerve activity in hypertension

The role of calcitonin gene-related peptide (CGRP)-containing vasodilator nerves in maintenance of hypertension was investigated in the perfused mesenteric vascular beds isolated from spontaneously hypertensive rats (SHR), deoxycorticosterone-salt-induced hypertensive rats (DOCA-Salt-HR) and corresponding normotensive control rats (Wistar Kyoto rats, WKY and Wistar rats, NR). In the mesenteric artery with an active tone, the neurogenic vasodilation induced by perivascular nerve stimulation (PNS, 0.5-8 Hz), which was mediated by CGRP nerves, was markedly decreased in adult SHR (15-week-old) when compared with age-matched WKY, whereas the vasodilation in DOCA-Salt-HR was similar in magnitude to that in NR. The vasodilator response to exogenously applied CGRP was greater in SHR than in WKY, whereas no difference was found between DOCA-Salt-HR and NR. The neurogenic release of CGRP-like immunoreactivity (CGRP-LI) induced by PNS of the mesenteric artery was significantly decreased in SHR compared to that of WKY. In addition, immunohistochemical studies showed decreased populations of CGRP-LI fibers in the mesenteric artery of SHR compared to those in WKY. These results suggest that CGRP-containing vasodilator innervation is greatly decreased in SHR with established hypertension. It is also suggested that the decreased vasodilator mechanism by CGRP-containing nerves contributes to the maintenance of hypertension.

Pressor response to microinjection of clonidine into the hypothalamic paraventricular nucleus in conscious rats

We have reported that intracerebroventricular (i.c.v.) injection of clonidine causes pressor response in conscious rats. To determine the effective brain site, cardiovascular responses induced by unilateral microinjection of clonidine into various hypothalamic nuclei of conscious rats were studied. Microinjection of clonidine (5-20 micrograms/0.5 microliter) into the paraventricular nucleus (PVN) of conscious rats dose-dependently produced a long-lasting pressor response with a decrease in heart rate, which mimicked the response to i.c.v. injection of clonidine. However, clonidine (10 micrograms) injection into various hypothalamic nuclei (anterior, posterior, ventromedial and dorsomedial nucleus) caused a small or no pressor response. In anesthetized rats, clonidine injected into the PVN induced a long-lasting depressor response concomitant with bradycardia. PVN pretreatment with the alpha 2-adrenoceptor antagonist, yohimbine (1 and 10 micrograms), dose-dependently inhibited the pressor response to PVN injected clonidine, but the alpha 1-adrenoceptor antagonist, prazosin (1 microgram), had no significant effect. Central (i.c.v.) pretreatment with the vasopressin (AVP) V1-receptor antagonist, [d(CH2)5Tyr(Me)]-AVP (0.5 and 2.0 micrograms), dose-dependently inhibited the pressor response to PVN injection of clonidine (10 micrograms), while systemic (i.v.) and local (intra-PVN injection) pretreatments with V1-receptor antagonist (2.0 micrograms) had no effect. These results suggest that the pressor response to microinjection of clonidine into the PVN of conscious rats is mediated by endogenous brain AVP, which is released by activation of alpha 2-adrenoceptors. It is also suggested that the PVN is a possible brain site for the pressor response to i.c.v. injected clonidine.

Age-related decrease of neurogenic release of calcitonin gene-related peptide from perivascular nerves in spontaneously hypertensive rats.

Changes in the neurogenic release of calcitonin gene-related peptide (CGRP) by perivascular nerve stimulation (PNS) were investigated in perfused mesenteric vascular beds isolated from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). CGRP-like immunoreactivity (CGRP-LI) in the perfusate was examined by radioimmunoassay (RIA) and identified to be CGRP itself by combined analysis with radioimmunoassay and HPLC. In SHR and WKY preparations, PNS (4 and 8 Hz) evoked a frequency-dependent increase of CGRP-LI in the perfusate, which was abolished by 500 nM tetrodotoxin and the removal of Ca2+ from the medium. In young SHR and WKY (8-week-old), there was no significant difference in the release of CGRP-LI induced by PNS, whereas the release in the 15-week-old SHR was significantly less than in the 15-week-old WKY and in 8-week-old SHR. These results suggest that neural release of CGRP from perivascular nerves in mesenteric resistance vessels of SHR decreases with age.

Presynaptic α2-adrenoceptor modulation of 5-hydroxytryptamine and noradrenaline release from vascular adrenergic nerves

Modulation of the stimulation-evoked release of 5-hydroxytryptamine (5-HT) and noradrenaline (NA) by presynaptic alpha 2-adrenoceptors was characterized in the perfused mesenteric vascular bed of the rat. The vasoconstrictor response to periarterial nerve stimulation (PNS; 8 Hz), previously abolished in the presence of 30 nM prazosin, was restored after 15 min treatment with 10 microM 5-HT, without a significant effect on the pressor response to 1 nmol of infused NA, which was previously abolished with prazosin. The restored pressor response to PNS was abolished by 100 nM tetrodotoxin and 100 nM ketanserin. Clonidine (1-10 microM) in the presence of prazosin induced a dose-dependent potentiation of the restored pressor response to PNS after 5-HT treatment while BHT 920 (10 nM-1 microM) and 100 nM clonidine inhibited the restored response. In the presence of 100 nM phentolamine, the restored pressor response to PNS was not altered by clonidine, but was inhibited by BHT 920. The PNS (8 Hz)-evoked tritium release in a preparation labeled with [3H]5-HT was facilitated by clonidine (100 nM-10 microM) while BHT 920 (10 nM-1 microM) and cocaine (1-10 microM) reduced the release. Yohimbine (1 microM) antagonized the effects of clonidine and cocaine but not of BHT 920 on the PNS-evoked tritium release. In the preparation labeled with [3H]NA, clonidine did not alter the PNS-evoked tritium release while BHT 920 inhibited it and cocaine facilitated it. Yohimbine did not antagonize the effect of BHT 920.(ABSTRACT TRUNCATED AT 250 WORDS)

Exaggerated pressor response to centrally administered renin in freely moving spontaneously hypertensive rats

The cardiovascular responses to intracerebroventricular (i.c.v.) injection of renin were compared between freely moving normotensive Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). The i.c.v. injection of renin (0.05-1.0 mU) produced a dose-dependent and a long-lasting rise in mean blood pressure associated with variable changes in heart rate (HR) in both WKY and SHR. However, the blood pressure and HR were not affected by intravenously injected renin (0.1 mU). The pressor response to i.c.v. injected renin was greater in SHR than in WKY, the dose-response curve for renin in SHR being to the left of that in WKY. Central (i.c.v.) pretreatment with [Sar1, Ile8]angiotensin II (50 micrograms) largely abolished the pressor response to i.c.v. injected renin in both WKY and SHR. The i.c.v. injection of angiotensin II (ANG II) (10-100 ng) induced a dose-dependent pressor response which was antagonized by central pretreatment with [Sar1, Ile8]ANG II (50 micrograms). The pressor response to ANG II was also greater in SHR than in WKY. These results suggest that the pressor response to centrally administered renin as well as to ANG II, which is mediated via ANG II receptors located in the brain, is enhanced in SHR.

Age-Related Changes in Calcitonin Gene-Related Peptide (CGRP)-Mediated Neurogenic Vasodilation of the Mesenteric Resistance Vessel in SHR

Age-related changes in CGRP-containing vasodilator nerve activity in hypertension were studied in perfused mesenteric vascular beds isolated from SHR and normotensive rats (WKY). Perivascular nerve stimulation (PNS; 0.5-8 Hz) of both SHR and WKY preparations with active tone produced a frequency-dependent vasodilator response, which was abolished by 100 nM tetrodotoxin, 500 nM capsaicin or cold storage denervation. This response in SHR greatly decreased with age, whereas the response in WKY slightly decreased with age. The neurogenic vasodilation in 15- and 30-week-old SHR but not 8-week-old SHR was significantly smaller than that in age-matched WKY. Vasodilator responses to exogenous CGRP (0.1-30 nM) in SHR increased with age, whereas an age-related decrease in the vasodilation was found in WKY. Immunohistochemical studies showed an age-related decrease in CGRP-containing fibers in SHR. These results suggest that CGRP-containing vasodilator innervation is greatly decreased when SHR develop and maintain hypertension.

Effect of angiotensin III (des-Asp1-angiotensin II) on the vascular adrenergic neurotransmission in spontaneously hypertensive rats

The effects of angiotensin III (des-Asp1-angiotensin II) on the pressor responses of the perfused mesenteric vascular bed to periarterial nerve stimulation (PNS) and exogenously administered noradrenaline (NA) of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) were compared. Angiotensin III (10, 20, 30 and 50 ng/ml) induced a marked potentiation of the pressor response to PNS (8 Hz) in a concentration-dependent manner with a slight elevation of the basal perfusion pressure in both SHR and WKY. The facilitatory effect of angiotensin III was blocked by [Sar1,Ile8]angiotensin II (200 ng/ml) and did not significantly differ for SHR and WKY. Angiotensin III also potentiated the pressor response to infusion of NA (50 ng) to the same extent in SHR and WKY. The degree of potentiation of the response to NA was similar to that to PNS in both WKY and SHR. Perfusion of angiotensin III (50 ng/ml) did not alter the increase in the 3H-efflux evoked by PNS (8 Hz) in the perfused mesenteric vascular bed prelabelled with [3H]NA, whereas the peptide potentiated significantly the pressor response to PNS in WKY and SHR to the same extent. These results suggest that angiotensin III postsynaptically facilitates the adrenergic neurotransmission of the mesenteric vascular bed to the same extent in WKY and SHR.