AT1 receptors mediate pressor responses induced by angiotensin II in the periaqueductal gray area of rats

Microinjection of angiotensin II (ANGII) (0.01 to 1 nmol) into the periaqueductal gray area (PAG) of anaesthetised rats caused dose-dependent increases in blood pressure. Preinjection (10 min before) of losartan (a selective non-peptide AT₁ receptor antagonist; 50 nmol) to the PAG reduced the pressor response to ANGII whereas PD123319 (a selective non-peptide AT₂ receptor antagonist; 50 nmol) did not affect the ANGII-induced hypertension. Thus, our data suggest that the activation of AT₁ but not AT₂ receptors mediates ANGII-induced blood pressure changes in the PAG area.     

A projection from the ventral tegmental area to the periaqueductal gray involved in cardiovascular regulation

Experiments were done in alpha-chloralose-anesthetized rats to determine a pathway mediating the cardiovascular depressor responses elicited from stimulation of the ventral tegmental area (VTA). The magnitude of the depressor responses elicited by glutamate stimulation (0.1 M/30 nl) of the VTA was examined after neuronal block produced by microinjections of lidocaine into ascending fiber bundles leaving the VTA to innervate the forebrain and thalamus. Bilateral microinjections of 1 microl of 4% lidocaine in the medial forebrain bundle (n = 6) and in the periventricular fibers of the midbrain (n = 5) did not attenuate the depressor response from stimulation of the VTA. Experiments were done using the anterograde tracer biotinylated dextran amine to identify descending projections from the VTA to cardiovascular centers in the brain stem. Examination of the nucleus of the solitary tract, ventrolateral medulla, and A5 catecholaminergic cell group revealed few or no fibers or terminals. Occasional fibers and some terminals were observed in the nucleus of raphe magnus, parabrachial nucleus, and locus ceruleus. A very dense bilateral projection was found to the ventrolateral periaqueductal gray (PAGvl) and dorsal raphe nucleus adjacent to the PAGvl. Bilateral injections of 4% lidocaine (n = 4) or 10 mM cobalt chloride (n = 5) into the PAGvl region attenuated the depressor responses elicited by stimulation of the VTA by approximately 50%. These experiments indicate that the depressor responses elicited from activation of the VTA are mediated in part by a pathway to a cardiovascular depressor area located in the PAGvl.     

Endothelin receptor antagonists reduce the pressor effects of angiotensin II into the periaqueductal gray area of rats.

Injection of ANGII (0.01, 0.1 and 1 nmol/rat) into the periaqueductal gray (PAG) area significantly (P<0.01) increased, in a dose-dependent manner, the mean arterial blood pressure (MAP). The increases in blood pressure induced by ANGII (1 nmol; 37 +/- 4 mmHg, n=5) were greatly reduced (>85%) by pre-administration of the ET(A) receptor antagonist FR139317 (5 nmol/rat) to the PAG area, but were unaffected by the ET(B) receptor antagonist BQ-788 (5 nmol/rat). SB209670, non-selective ET(A)/ET(B) receptor antagonist, also reduced the effect induced by ANGII. These results suggest that endogenous endothelin-1, via an action on ET(A) receptors, may contribute to the pressor effects of ANGII within the PAG area of rats.     

Protective role of ETA endothelin receptors during the acute phase of Trypanosoma cruzi infection in rats

Chagas' disease, caused by Trypanosoma cruzi, has an acute phase characterized by blood-circulating trypomastigotes and amastigote proliferation in several cell types, especially muscle cells. In the chronic phase, around 70% of infected people are asymptomatic (latent form). The remainder develop chagasic cardiomyopathy and/or digestive syndromes. There is evidence for aggravation of the chronic cardiac pathology by endothelin-mediated vasoconstriction. Holtzman rats have proven to be a good model for Chagas' disease acute phase and latent chronic phase. Now, we investigate the effects of prolonged treatment with an endothelin ET(A) receptor antagonist, BSF 461314, during the acute phase on parasitemia, coronary flow, tissue parasitism and the inflammatory process. Using isolated heart in Langendorff's preparation, endothelial dysfunction was observed only in non-treated infected animals. Histoquantitative analyses carried out in heart and diaphragm showed higher tissue parasitism and/or inflammatory process in BSF 461314-treated animals. Our data indicate that endothelin ET(A) receptors contribute to the initial mechanisms of parasite control. Impairment of the endothelium-dependent vasodilatation favors hazardous effects. However, blocking endothelin ET(A) receptors can prevent the latter.     

The diagonal band of Broca is involved in the pressor pathway activated by noradrenaline microinjected into the periaqueductal gray area of rats

AimsThe dorsal periaqueductal gray area (dPAG) is involved in cardiovascular modulation. Previously, we reported that noradrenaline (NA) microinjection into the dPAG caused a pressor response that was mediated by vasopressin release into the circulation. However, the neuronal pathway that mediates this response is as yet unknown. There is evidence that chemical stimulation of the diagonal band of Broca (dbB) also causes a pressor response mediated by systemic vasopressin release. In the present study, we evaluated the participation of the dbB in the pressor response caused by NA microinjection into the dPAG as well as the existence of neural connections between these areas.Main methodsWith the above goal, we verified the effect of the pharmacological ablation of the dbB on the cardiovascular response to NA microinjection into the dPAG of unanesthetized rats. In addition, we microinjected the neuronal tracer biotinylated-dextran-amine (BDA) into the dPAG and looked for efferent projections from the dPAG to the dbB.Key findingsThe pharmacologically reversible ablation of the dbB with local microinjection of CoCl₂ significantly reduced the pressor response caused by NA microinjection (15 nmol/50 nL) into the dPAG. In addition, BDA microinjection into the dPAG labeled axons in the dbB, pointing to the existence of direct connections between these areas.SignificanceThe present results indicate that synapses within the dbB are involved in the pressor pathway activated by NA microinjection into the dPAG and direct neural projection from the dPAG to the dbB may constitute the neuroanatomic substrate for this pressor pathway.     

Effects of L-NAME on endothelin-1-induced barrel-rolling in periaqueductal gray area of rats

The injection of endothelin-1 (ET-1) into the dorsolateral periaqueductal gray (PAG) area of freely moving rats at doses from 0.1 to 1 pmol/rat induced rotation along the long axis of the body (barrel-rolling). The pretreatment of this area with L-NAME (Nω-nitro-L-arginine methyl ester, 1 μmol/rat), an L-arginine analogue and a potent inhibitor of nitric oxide (NO) biosynthesis, significantly (p< 0.01) potentiated the duration of the ET-1-induced barrel-rolling. Pretreatment of the PAG area with L-arginine (1 μmol/rat), a precursor of NO, significantly (p < 0.01) decreased the ET-1-induced effects. These preliminary data indicate that the L-arginine-NO pathway exerts a functional antagonism on ET-1 induced barrel-rolling at the level of the PAG area.     

Role of endothelin (ETA) receptors in neonatal morphine withdrawal

We have previously demonstrated role of central endothelin (ET) receptors in neonatal morphine tolerance. The present study was conducted to investigate involvement of central ET receptors in neonatal rat morphine withdrawal. The aim was to determine activation of G-proteins coupled to opioid and ET receptors by morphine and ET ligands in neonatal rat brains during morphine withdrawal. Pregnant female rats were rendered tolerant to morphine by chronic exposure to morphine pellets over 7 days. Withdrawal was induced on day 8 by removal of pellets. Rat pups were delivered by cesarean section 24 h after pellet removal. G-protein stimulation induced by morphine; ET-1; ETA receptor antagonist, BMS182874; and ETB receptor agonist, IRL1620, was determined in the brain of neonatal rats undergoing morphine withdrawal by [35S]GTPgammaS binding assay. Morphine-induced maximal stimulation of G-protein in morphine withdrawal group (83.60%) was significantly higher compared to placebo control group (66.81%). EC50 value for ET-1-induced G-protein stimulation during morphine withdrawal (170.60 nM) was higher than control (62.5 nM). BMS182874, did not stimulate GTP binding in control but significantly increased maximal stimulation of G-proteins in morphine withdrawal (86.07%, EC50 = 31.25 nM). IRL1620-induced stimulation of G-proteins was similar in control and morphine withdrawal. The present findings indicate involvement of central ETA receptors in neonatal morphine withdrawal.     

ETA receptor-mediated role of endothelin in the kidney of doca-salt hypertensive rats

Renal effects of FR139317, an endothelin ET_A receptor antagonist, were examined using anesthetized normotensive and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The intravenous bolus injection of FR139317 (10 mg/kg) produced a slight decrease in mean blood pressure (MAP; −13%) in the control rats and this hypotension was accompanied by a moderate renal vasodilation (renal vascular resistance: RVR; −12%). In the DOCA-salt hypertensive rat, FR139317 had a more pronounced hypotensive effect (MAP; −26%) accompanied by a potent renal vasodilation (RVR; −33%). FR 139317 significantly increased renal blood flow only in the DOCAsalt rats. In contrast, FR139317 produced a significant decrease in urine flow and urinary sodium excretion only in control rats. Northern blot analysis revealed that the renal prepro endothelin-1 (ET-1) mRNA level was significantly increased in DOCA-salt hypertensive rats. Thus, it seems likely that endogenous ET-1 is responsible for the maintenance of DOCA-salt-induced hypertension. We also suggest that at least in part, ET-1 and £ta receptors are involved in renal hemodynamic abnormalities in DOCA-salt-induced hypertension. The augmentation of renal ET-1 production may possibly have a function in the development and maintenance of DOCA-salt-induced hypertension.     

Characterization of endothelin receptors ETA and ETB expressed in COS cells.

The cloned cDNA genes for endothelin receptors ETA and ETB were expressed in COS cells, and the binding characteristics of the two receptors with three isopeptide ligands (ET-1, ET-2, and ET-3) were examined in detail. The results indicated that the stability of receptor-ET-1 complexes formed with ETA and ETB were significantly different from each other, while their affinities to ET-1 were similar. The preformed ETA-ET-1 complex readily dissociated upon SDS-PAGE, as did many of the other receptors so far studied, while the ETB-ET-1 complex survived SDS-PAGE when it was run at low temperature (approximately 4 degrees C). Clear differences in stability were also shown in comparative studies of acid treatment of the two types of complexes. Only the ETB-ET-1 complex was resistant to acid treatment (0.2 M acetic acid, 0.5 M NaCl), and its 50 kDa monomeric receptor-ligand complex remained intact. The ETB-ET-1 complex (50 kDa) formed at 4 degrees C on the surface of COS cells, however, was susceptible to limited proteolysis at 37 degrees C that reduced the molecular size of the complex to a distinct 35 kDa. No such size reduction was observed with the preformed ETA-ET-1 complex. The overall structure of two endothelin receptors, as deduced from the sequence of cloned cDNAs, is similar in many respects. However, the present findings demonstrate distinct differences in the biochemical nature of the two receptors, which suggest their distinct biological functions.     

A visual circuit related to the periaqueductal gray area for the antinociceptive effects of bright light treatment

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Participation of midbrain periaqueductal gray matter in defense conditioning in rats

Responses of neurons of the periaqueductal gray matter (PAG) were studied in chronic experiments on cats during formation and extinction of a defensive conditioned reflex to sound and its differential inhibition. In response to conditioned stimulation these neurons developed phasic-tonic spike responses up to 3 sec in duration. During combination of stimuli these responses were formed long before the conditioned reflex and disappeared long after the latter was extinguished. In the case of an established conditioned reflex, the onset of spike responses occurred 100–200 msec before the appearance of motor responses. An increase in spike activity of tonic character in neurons of PAG preceded voluntary movements by 100–500 msec. The responses of these neurons to presentation of a differential stimulus consisted of groups of spikes 150–200 msec in duration. They were formed with difficulty, and their manifestation was made even more difficult by an interruption during the experiment and by preceding positive stimuli. On the basis of the results a conditioned reflex can be regarded as the result of a multilevel hierarchic process of readjustment of unit activity, which begins in the nonspecific structures of the midbrain.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 15, No. 3, pp. 278–287, May–June, 1983.     

CRF type 1 receptors in the dorsal periaqueductal gray modulate anxiety-induced defensive behaviors

The dorsal periaqueductal gray (dPAG) is involved in defensive coping reactions to threatening stimuli. Corticotropin releasing factor (CRF) is substantially implicated as a direct modulator of physiological, endocrine and behavioral responses to a stressor. Previous findings demonstrate a direct role of the central CRF system in dPAG-mediated defensive reactions toward a threatening stimulus. These include anxiogenic behaviors in the elevated plus maze (EPM) in rats and defensive reactions in both the mouse defense test battery (MDTB) and rat exposure test (RET) paradigms in mice. Furthermore, CRF was shown to directly and dose-dependently excite PAG neurons in vitro. The aim of the present series of experiments was to directly evaluate the role of the CRF1 receptor (CRF1) in dPAG-induced defensive behaviors in the MDTB and the RET paradigms. For this purpose, cortagine, a novel CRF1-selective agonist, was directly infused into the dPAG. In the RET the high dose of cortagine (100 ng) significantly affected spatial avoidance measures and robustly increased burying behavior, an established avoidance activity, while having no effects on behaviors in the MDTB. Collectively, these results implicate CRF1 in the dPAG as a mediator of temporally and spatially dependent avoidance in response to controllable and constant stimuli.     

RGS14 prevents morphine from internalizing Mu-opioid receptors in periaqueductal gray neurons

Opioid agonists display different capacities to stimulate mu-opioid receptor (MOR) endocytosis, which is related to their ability to provoke the phosphorylation of specific cytosolic residues in the MORs. Generally, opioids that efficiently promote MOR endocytosis and recycling produce little tolerance, as is the case for [d-Ala², N-MePhe⁴,Gly-ol⁵] encephalin (DAMGO). However, morphine produces rapid and profound antinociceptive desensitization in the adult mouse brain associated with little MOR internalization. The regulator of G-protein signaling, the RGS14 protein, associates with MORs in periaqueductal gray matter (PAG) neurons, and when RGS14 is silenced morphine increased the serine 375 phosphorylation in the C terminus of the MOR, a GRK substrate. Subsequently, these receptors were internalized and recycled back to the membrane where they accumulated on cessation of antinociception. These mice now exhibited a resensitized response to morphine and little tolerance developed. Thus, in morphine-activated MORs the RGS14 prevents GRKs from phosphorylating those residues required for β-arresting-mediated endocytosis. Moreover morphine but not DAMGO triggered a process involving calcium/calmodulin-dependent kinase II (CaMKII) in naïve mice, which contributes to MOR desensitization in the plasma membrane. In RGS14 knockdown mice morphine failed to activate this kinase. It therefore appears that phosphorylation and internalization of MORs disrupts the CaMKII-mediated negative regulation of these opioid receptors.     

ETA and ETB receptors are expressed in vascular adventitial fibroblasts

The adventitia has been recognized to play important roles in vascular oxidative stress, remodeling, and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin (ET)-1 in response to ANG II. However, it is unclear whether ET-1 receptors are expressed in the adventitia. We therefore investigated the expression and roles of both ET(A) and ET(B) receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Adventitial fibroblasts were isolated and cultured from the mouse thoracic aorta by the explant method. Cultured cells were treated with ANG II (100 nmol/l) or ET-1 (10 pM) in the presence or absence of the ANG II type 1 receptor antagonist losartan (100 μM), the ET-1 receptor antagonists BQ-123 (ET(A) receptor, 1 μM) and BQ-788 (ET(B) receptor, 1 μM), and the ET(B) receptor agonist sarafotoxin 6C (100 nM). ET-1 peptide levels were determined by ELISA, whereas ET(A), ET(B), and collagen levels were determined by Western blot analysis. ANG II increased ET-1 peptide levels in a time-dependent manner. ANG II increased ET(A) and ET(B) receptor protein levels as well as collagen in a similar fashion. ANG II-induced collagen was reduced while in the presence of BQ-123, suggesting a role for the ET(A) receptor in the regulation of the extracellular matrix. ANG II treatment in the presence of BQ-788 significantly increased ET-1 peptide levels. Conversely, the ET(B) receptor agonist sarafotoxin 6C significantly decreased ET-1 peptide levels. These data implicate a role for the ET(B) receptor in the clearance of the ET-1 peptide. In conclusion, both ET(A) and ET(B) receptors are expressed in adventitial fibroblasts, which paves the ground for the biological significance of adventitial ET-1. The ET(A) receptor subtype mediates collagen I expression, whereas the ET(B) receptor subtype may play a protective role through increasing the clearance of the ET-1 peptide.     

Respiratory muscle responses elicited by dorsal periaqueductal gray stimulation in rats

The periaqueductal gray matter is an essential neural substrate for central integration of defense behavior and accompanied autonomic responses. The dorsal half of the periaqueductal gray matter (dPAG) is also involved in mediating emotional responses of anxiety and fear, psychological states that often are associated with changes in ventilation. However, information regarding respiratory modulation elicited from this structure is limited. The present study was undertaken to investigate the relationship between stimulus frequency and magnitude on ventilatory pattern and respiratory muscle activity in urethane-anesthetized, spontaneously breathing rats. Electrical stimulation in the dPAG-recruited abdominal muscle activity increased ventilation and increased respiratory frequency by significantly shortening both inspiratory time and expiratory time. Ventilation increased within the first breath after the onset of stimulation, and the respiratory response increased with increasing stimulus frequency and magnitude. dPAG stimulation also increased baseline EMG activity in the diaphragm and recruited baseline external abdominal oblique EMG activity, normally quiescent during eupneic breathing. Significant changes in cardiorespiratory function were only evoked by stimulus intensities >10 microA and when stimulus frequencies were >10 Hz. Respiratory activity of both the diaphragm and abdominal muscles remained elevated for a minimum of 60 s after cessation of stimulation. These results demonstrate that there is a short-latency respiratory response elicited from the dPAG stimulation, which includes both inspiratory and expiratory muscles. The changes in respiratory timing suggest rapid onset and sustained poststimulus dPAG modulation of the brain stem respiratory network that includes expiratory muscle recruitment.     

Cellular action of cholecystokinin-8S-mediated excitatory effects in the rat periaqueductal gray

The peptide cholecystokinin (CCK) is one of the major neurotransmitters modulating satiety, nociception, and anxiety behavior. Although many behavioral studies showing anti-analgesic and anxiogenic actions of CCK have been reported, less is known about its cellular action in the central nervous system (CNS). Therefore, we examined the action of CCK in rat dorsolateral periaqueductal gray (PAG) neurons using slice preparations and whole-cell patch-clamp recordings. Application of CCK-8S produced an inward current accompanied by increased spontaneous synaptic activities. The CCK-8S-induced inward current (I(CCK)) was recovered after washout and reproduced by multiple exposures. Current-voltage plots revealed that I(CCK) reversed near the equilibrium potential for K(+) ions with a decreased membrane conductance. When several K(+) channel blockers were used, application of CdCl(2), TEA, or apamin significantly reduced I(CCK). I(CCK) was also significantly reduced by the CCK(2) receptor antagonist, L-365,260, while it was not affected by the CCK(1) receptor antagonist, L-364,718. Furthermore, we examined the effects of CCK-8S on miniature excitatory postsynaptic currents (mEPSCs) in order to determine the mechanism of CCK-mediated increase on synaptic activities. We found that CCK-8S increased the frequency of mEPSCs, but had no effect on mEPSC amplitude. This presynaptic effect persisted in the presence of CdCl(2) or Ca(2+)-free bath solution, but was completely abolished by pre-treatment with BAPTA-AM, thapsigargin or L-365,260. Taken together, our results indicate that CCK can excite PAG neurons at both pre- and postsynaptic loci via the activation of CCK(2) receptors. These effects may be important for the effects of CCK on behavior and autonomic function that are mediated via PAG neurons.     

Respiratory response to activation or disinhibition of the dorsal periaqueductal gray in rats.

The neural substrates mediating autonomic components of the behavioral defense response have been shown to reside in the periaqueductal gray (PAG). The cardiovascular components of the behavioral defense response have been well described and are tonically suppressed by GABAergic input. The ventilatory response associated with disinhibition of the dorsal PAG (dPAG) neurons is unknown. In urethane-anesthetized, spontaneously breathing rats, electrical stimulation of the dPAG was shown to decrease the expiration time and increase respiratory frequency, with no change in time of inspiration. Baseline and the change in diaphragm electromyograph also increased, resulting in an increase in neural minute activity. Microinjection of bicuculline methobromide, a GABA(A)-receptor antagonist, into the dPAG produced a similar response, which was dose dependent. Disinhibition of the dPAG also produced a decrease in inspiration time. These results suggest that GABA(A)-mediated suppression of dPAG neurons plays a role in the respiratory component of behavioral defense responses. The respiratory change is due in part to a change in brain stem respiratory timing and phasic inspiratory output. In addition, there is an increase in tonic diaphragm activity.     

Role of serotonin receptors in panic-like behavior induced by nitric oxide in the rat dorsolateral periaqueductal gray: effects of chronic clomipramine treatment

Local administration of serotonin (5-HT) receptor agonists inhibits panic-like reactions induced by electrical stimulation of the rat dorsolateral periaqueductal grey (dlPAG). This anti-aversive effect is enhanced by chronic treatment with anti-panic drugs such as clomipramine. Since nitric oxide (NO) may mediate panic-like behavior in the dlPAG, we tested the hypothesis that chronic clomipramine treatment would also potentiate the effects of locally injected 5-HT-receptor agonists on panic-like reactions induced by intra-dlPAG injection of an NO-donor (SIN). After 21 days of daily i.p. injections of saline or clomipramine (10 mg/kg) male Wistar rats received local injections of saline, the 5-HT(1A)-receptor agonist 8-OH-DPAT (8 nmol) or the 5-HT2A/2C-receptor agonist DOI (16 nmol) followed by saline or SIN (150 nmol). NO-induced panic-like reactions were inhibited by DOI, but not by 8-OH-DPAT. Chronic clomipramine did not modify these effects but tended to produce anti-aversive effect by itself. In chronically clomipramine treated animals 8-OH-DPAT potentiated NO-induced panic-like reactions. The results indicate that the panic-like effects of NO in the dlPAG may be attenuated by 5-HT2A/2C-, but not by 5-HT1A-receptors. The anti-aversive effect of DOI is not modified by chronic clomipramine treatment.     

Glutamate release in midbrain periaqueductal gray by activation of skeletal muscle receptors and arterial baroreceptors

We have previously reported that both skeletal muscle receptor and arterial baroreceptor afferent inputs activate neurons in the dorsolateral (DL) and lateral regions of the midbrain periaqueductal gray (PAG). In this study, we determined whether the excitatory amino acid glutamate (Glu) is released to mediate the increased activity in these regions. Static contraction of the triceps surae muscle for 4 min was evoked by electrical stimulation of the L7 and S1 ventral roots in cats. Activation of arterial baroreceptor was induced by intravenous injection of phenylephrine. The endogenous release of Glu from the PAG was recovered with the use of a microdialysis probe. Glu concentration was measured by the HPLC method. Muscle contraction increased mean arterial pressure (MAP) from 98 +/- 10 to 149 +/- 12 mmHg (P < 0.05) and increased Glu release in the DL and lateral regions of the middle PAG from 0.39 +/- 0.10 to 0.73 +/- 0.12 microM (87%, P < 0.05) in intact cats. After sinoaortic denervation and vagotomy were performed, contraction increased MAP from 95 +/- 12 to 158 +/- 15 mmHg, and Glu from 0.34 +/- 0.08 to 0.54 +/- 0.10 microM (59%, P < 0.05). The increases in arterial pressure and Glu were abolished by muscle paralysis. Phenylephrine increased MAP from 100 +/- 13 to 162 +/- 22 mmHg and increased Glu from 0.36 +/- 0.10 to 0.59 +/- 0.18 microM (64%, P < 0.05) in intact animals. Denervation abolished this Glu increase. Summation of the changes in Glu evoked by muscle receptor and arterial baroreceptor afferent inputs was greater than the increase in Glu produced when both reflexes were activated simultaneously in intact state (123% vs. 87%). These data demonstrate that activation of skeletal muscle receptors evokes release of Glu in the DL and lateral regions of the middle PAG, and convergence of afferent inputs from muscle receptors and arterial baroreceptors in these regions inhibits the release of Glu. These results suggest that the PAG is a neural integrating site for the interaction between the exercise pressor reflex and the arterial baroreceptor reflex.