Tonic suppression of baroreceptor reflex by endogenous neurotensin in the rat

We evaluated the modulatory role of endogenous neurotensin (NT) in baroreceptor reflex (BRR) response in Sprague-Dawley rats anesthetized with pentobarbital sodium. Intracerebroventricular (i.c.v.) administration of NT (15 or 30 nmol) significantly reduced the sensitivity of the BRR response. Blocking the endogenous activity of the tridecapeptide with its specific antagonist, (D-Trp11)-NT (4 or 8 nmol) or antiserum against NT (1:20); or inhibiting the aminopeptidases with bestatin (200 nmol), on the other hand, promoted a potentiation of BRR response. When administered together with bestatin (200 nmol), the suppressive effect of NT (15 nmol) on the BRR response was further enhanced, as was the augmentative action of (D-Trp11)-NT (4 nmol). Upon microinjection into the bilateral nucleus tractus solitarius (NTS), NT (600 pmol) and (D-Trp11)-NT (150 pmol) respectively elicited a reduction and enhancement of the BRR response. These results suggest that neurons that contain NT may participate in central cardiovascular regulation by tonically suppressing the BRR, possibly via an action on the NTS where baroreceptor afferents terminate.     

Facilitation of baroreceptor reflex response by endogenous somatostatin in the rat.

We evaluated the potential participation of endogenous brain somatostatin-14 (SOM) in central cardiovascular regulation, using adult male Sprague-Dawley rats anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Intracerebroventricular (i.c.v.) application of SOM (2 or 4 nmol) promoted a significant elevation in baroreceptor reflex (BRR) response, induced by phenylephrine (5 micrograms kg, i.v.). Blocking the endogenous SOM activity with its specific receptor antagonist, cyclo-[7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)] (2 or 4 nmol, i.c.v.) or antiserum against SOM (1:20, i.c.v.), on the other hand, appreciably attenuated the same response. These modulatory effects on the BRR response were essentially duplicated upon bilateral microinjections of SOM (320 pmol), SOM antagonist (320 pmol) or anti-SOM (1:20) into the caudal portion of the nucleus of tractus solitarius (NTS), the terminal site for baroreceptor afferents. These results suggest that neurons that contain SOM may participate in cardiovascular control by tonically facilitating the BRR, possibly by exerting an influence on the neurons at the NTS.     

Substance P in the baroreceptor reflex: 25 years

Twenty-five years ago, very little was known about chemical communication in the afferent limb of the baroreceptor reflex arc. Subsequently, considerable anatomic and functional data exist to support a role for the tachykinin, substance P (SP), as a neuromodulator or neurotransmitter in baroreceptor afferent neurons. Substance P is synthesized and released from baroreceptor afferent neurons, and excitatory SP (NK1) receptors are activated by baroreceptive input to second-order neurons. SP appears to play a role in modulating the gain of the baroreceptor reflex. However, questions remain about the specific role and significance of SP in mediating baroreceptor information to the central nervous system (CNS), the nature of its interaction with glutaminergic transmission, the relevance of colocalized agents, and complex effects that may result from mediation of non-baroreceptive signals to the CNS.     

5-HT-mediated inhibition of cardiovagal baroreceptor reflex response during defense reaction in the rat

Previous studies showed that the cardiac response of the baroreceptor reflex (bradycardia) is inhibited during the defense reaction evoked by direct electrical or chemical stimulation of the periaqueductal gray (dPAG) in the rat. Whether central serotonin and nucleus tractus solitarius (NTS) serotonin(3) (5-HT(3)) receptors might participate in this inhibition was investigated in urethane-anesthetized and atenolol-pretreated rats. Our results showed that both electrical and chemical stimulation of the dPAG produced a drastic reduction of the cardiovagal component of the baroreceptor reflex triggered by either intravenous administration of phenylephrine or aortic nerve stimulation. This inhibitory effect of dPAG stimulation on the baroreflex bradycardia was not observed in rats that had been pretreated with p-chlorophenylalanine (300 mg/kg ip daily for 3 days) to inhibit serotonin synthesis. Subsequent 5-hydroxytryptophan administration (60 mg/kg ip), which was used to restore serotonin synthesis, allowed the inhibitory effect of dPAG stimulation on both aortic and phenylephrine-induced cardiac reflex responses to be recovered in p-chlorophenylalanine-pretreated rats. On the other hand, in nonpretreated rats, the inhibitory effect of dPAG stimulation on the cardiac baroreflex response could be markedly reduced by prior intra-NTS microinjection of granisetron, a 5-HT(3) receptor antagonist, or bicuculline, a GABA(A) receptor antagonist. These results show that serotonin plays a key role in the dPAG stimulation-induced inhibition of the cardiovagal baroreceptor reflex response. Moreover, they support the idea that 5-HT(3) and GABA(A) receptors in the NTS contribute downstream to the inhibition of the baroreflex response caused by dPAG stimulation.     

Participation of galaninergic neurotransmission at the paraventricular hypothalamic nucleus in the suppression of baroreceptor reflex response by locus ceruleus in the rat

We evaluated the potential participation of galanin (GAL) at the paraventricular nucleus of hypothalamus (PVN) in the suppression of baroreceptor reflex (BRR) response by locus ceruleus (LC), using adult male Sprague-Dawley rats anesthetized with pentobarbital sodium. Microinjection of GAL (100 pmol) bilaterally into the PVN significantly depressed the BRR response. This suppressive effect was appreciably antagonized when GAL (100 pmol) and GAL antiserum (1:20) were coadministered into the bilateral PVN. Whereas bilateral microinjection of GAL antiserum into the PVN by itself elicited minimal effect, it nevertheless significantly attenuated the suppressive effect of either electrical or chemical activation of LC on the BRR response. Pretreatment with the same amount of normal rabbit serum (1:20), on the other hand, was ineffective. These results suggest that a galaninergic projection from the LC to PVN may participate in the suppression of BRR response by this dorsal pontine nucleus.     

Participation of pertussis toxin-sensitive GTP-binding regulatory proteins in the suppression of baroreceptor reflex by neurotensin in the rat.

We evaluated the molecular mechanism that may underlie the suppressive effect of neurotensin (NT) on the baroreceptor reflex (BRR), using Sprague-Dawley rats that were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). Intracerebroventricular (i.c.v.) application of NT (15 nmol) significantly inhibited the BRR response. Such an inhibition was appreciably antagonized by pretreating animals with i.c.v. injection of pertussis toxin (10 or 20 pmol), N-ethylmaleimide (1 or 2 nmol), forskolin (30 or 60 nmol) or phorbol 12-myristate 13-acetate (2 or 4 nmol), but not by cholera toxin (15 or 30 pmol). More specifically, pretreatments with bilateral microinjection into the nucleus tractus solitarius (NTS) of pertussis toxin (80 or 160 fmol), N-ethylmaleimide (80 pmol), forskolin (480 pmol) or phorbol 12-myristate 13-acetate (16 or 32 pmol) also blunted the NT-induced suppression of BRR, although cholera toxin (120 or 240 fmol), or 1,9-dideoxyforskolin (480 pmol) had no appreciable effect. These results suggest that a pertussis toxin-sensitive guanine nucleotide-binding regulatory protein(s), which is not likely to be Gs, possibly Gi or Gp, may be involved in the transmembrane signaling process that underlies the suppression of BRR response by NT at the NTS.     

Inflammatory responses induced by substance P in rat paw.

Substance P (SP) injection in the plantar region of rat hind paw caused a dose related inflammation, which reached a peak within 10 min of injection and declined after 60 min. Low doses (0.25-0.063 mg/kg) of SP-antagonists like (D-Pro2, D-Trp7,9)-SP and (D-Pro2, D-Phe7, D-Trp9)-SP pretreatment significantly inhibited the SP induced paw oedema, while higher doses (0.5-1 mg/kg) showed agonistic effects. Pretreatment with diphenhydramine alone or along with low doses of SP-antagonists was highly significant in blocking this inflammation, the latter combination being more effective than the former. Pretreatment with acute capsaicin produced a synergestic effect on SP induced paw oedema, while pretreatment with chronic capsaicin significantly inhibited this SP induced paw oedema. The results indicate involvement of histamine and possible therapeutic importance of capsaicin in SP mediated inflammatory type of responses.     

Renal inactivation of substance P in the rat.

The activity and distribution of substance P-catabolizing enzyme(s) were studied in the rat kidney. Kidney homogenates inactive substance P 5-20 times as fast as do homogenates of intestine, liver, lung, heart or brain. The catabolizing activity was highest in the cortex and decreased progressively down the papilla. Cortex of rat kidney was homogenized and fractions enriched in microsomal membrane, final supernatant, plasma membrane, endoplasmic reticulum, brush border and intact glomeruli were prepared. The identity and homogeneity of the preparations were determined by assaying marker enzymes and by morphological examination. Substance P was catabolized most rapidly by the microsomal and plasma-membrane-enriched fractions, and least rapidly by endoplasmic reticulum or final supernatant fractions. Purified brush border of proximal tubules inactivated substance P more than 10 times as fast as isolated glomeruli. Our experiments show that substance P is catabolized at a rate that is similar to the rates of inactivation of bradykinin and angiotensin II. Further, the distribution of substance P-catabolizing activity in various kidney fractions is similar to the distribution of kininase and angiotensinase activities previously reported.     

Cholinergic neurons and the cardiovascular response produced by central injection of substance P in the normotensive rat

The role of cholinergic neurons in central cardiovascular regulation is not well understood, however, activation of brain cholinergic neurons in several species evokes a hypertensive response. As with central cholinergic stimulation, intracerebroventricular (i.c.v.) injection of substance P (sP) elicits a pressor response in unanesthetized rats. The purpose of this study was to determine whether the cardiovascular effects following i.c.v. injection of this neuropeptide are mediated by central cholinergic neurons. Therefore, the cardiovascular response to sP was examined in control rats, and in animals pretreated centrally with classical pre- and post-synaptic cholinergic antagonists. Drugs were administered directly into the lateral ventricle while rats were freely-moving in their home cages. I.c.v. injection of sP produced a dose - dependent increase in arterial pressure and heart rate. The hypertensive response was significantly reduced by pretreatment with hemicholinium-3. This dose (20 ug) of hemicholinium-3 is capable of producing a maximal depletion of brain acetylcholine levels. The increase in heart rate to substance P was not as sensitive to hemicholinium-3 pretreatment as was blood pressure. Central pretreatment with the nicotinic receptor antagonist, hexamethonium was more effective than the muscarinic antagonist, atropine in blocking the pressor response to sP. Hexamethonium did not significantly alter the tachycardic response to the peptide, but atropine produced a small, but significant reduction in the response. Therefore, the pressor response to central injection of sP may be mediated to a large extent through cholinergic pathways involving nicotinic receptors.     

Intrathecal cholecystokinin interacts with morphine but not substance P in modulating the nociceptive flexion reflex in the rat

The effect of intrathecal (IT) cholecystokinin (CCK), substance P (SP) and morphine (MO) on spinal cord excitability was studied in decerebrate, spinalized rats. CCK had a weaker facilitatory effect on the nociceptive flexion reflex than SP. The possible functional significance of the coexistence of CCK and SP in neurons projecting to the spinal cord was tested by coadministration of the two peptides. At the doses tested no synergistic interaction on the reflex was found with CCK and SP. IT MO caused a brief enhancement followed by a prolonged depression of the reflex. A high dose of CCK injected prior to MO increased the facilitatory effect and decreased the depressive effect of the opiate on the reflex. The effect of desulfated (D) CCK was similar to CCK but at a higher dose. Naloxone (NAL) had a similar effect as CCK when administered prior to MO. The MO-induced depression of the reflex was readily reversed by NAL, but not by CCK. The results indicate that CCK may prevent the inhibitory effect of MO on spinal cord excitability to nociceptive stimulation, but does not reverse it. CCK may alter the balance of excitation-inhibition between various types of dorsal horn interneurons that are involved in the transmission of nociceptive information.     

Analysis of the inflammatory response induced by substance P in the mouse pleural cavity.

This study analyzes both cell migration and exudation responses elicited by substance P (SP) in the mouse pleural cavity. SP caused, 4 h after its administration into the mouse pleural cavity, a dose-related recruitment of leukocytes (ED50 = 14.2 nmol), mainly due to mononuclears. Leukocytes peaked between 2 and 4 h, being followed by a slight decay that remained elevated for up to 24 h. Exudation, although small, was significantly elevated from 2 to 96 h after. NK1 (FK 888) or NK3 (SR 142801), but not NK2 (SR 48968) tachykinin receptor antagonists, significantly inhibited cell migration. HOE 140 and NPC 17731, bradykinin B2 receptor antagonists, caused graded inhibition of cell influx (ID50s of 0.03 and 0.04 pmol), but des-Arg9-Leu8-BK, B1 receptor antagonist, had no effect. The nitric oxide inhibitors L-NOARG and L-NAME, but not D-NAME, significantly inhibited SP-induced pleurisy. Pretreatment of the animals with indomethacin, dexamethasone, terfenadine, theophylline or salbutamol produced significant inhibition of the inflammatory parameters, whereas cromolyn only inhibited exudation. These results indicate that intrapleural injection of SP in mice elicit a long-lasting inflammatory reaction that is characterized by the participation of nitric oxide, kinins, cyclooxygenase metabolites and histamine. Antiasthmatic drugs such as theophylline, salbutamol, dexamethasone, and, to a lesser extent cromolyn, also markedly inhibit this inflammatory reaction. These results provide clear evidence supporting the role played by SP in neurogenic inflammation.     

Effect of a substance P antagonist on capsaicin-induced nociceptive reflex in the isolated spinal cord-tail preparation of the rat

An isolated spinal cord-tail preparation of the newborn rat was developed and used for studying the effects of various drugs. The cord and the tail were separately perfused with oxygenated artificial cerebrospinal fluid. Application of capsaicin in a small amount to the tail induced a depolarizing response of the lumbar ventral root (L3-L5) lasting for about 30 sec. The stimulating action of capsaicin was potentiated by previous perfusion of the tail with a medium containing prostaglandin E1 or E2. The capsaicin-induced nociceptive reflex was depressed by application to the spinal cord of morphine, Met-enkephalin, dynorphin (1-13), somatostatin, adenosine, GABA and a substance P (SP) antagonist [D-Arg1, D-Pro2, D-Trp7,9, Leu11]SP, and potentiated by bicuculline. The present preparation will be useful for the future studies on pain and analgesic drugs.     

N-terminally extended substance P is released together with substance P from rat spinal cord.

The release of different forms of substance P-like immunoreactivity (SP-LI) from superfused slices of rat spinal cord was studied. The released SP-LI was characterized by reverse-phase high-performance liquid chromatography and radioimmunoassay with two antisera directed to the C- and N-terminal parts of SP, respectively. The SP-LI detected in the superfusates with the C-terminally directed antiserum was found to consist of (undeca) SP, SP-sulfoxide and a late eluting component which was not detectable with the N-terminally directed antiserum. This component was also found in neutral extracts of the spinal cord. Upon trypsin digestion, it produced SP-LI detectable with both C- and N-terminally directed antiserum which also coeluted with SP. From these results we conclude that this form of SP-LI most likely corresponds to an N-terminally extended form of SP. An increase of the potassium concentration in the superfusion fluid from 5 to 50 mM evoked an increased overflow of both SP and the N-terminally extended SP. The present results indicate that N-terminally extended SP is released by a calcium-dependent mechanism together with SP from terminals in the spinal cord in response to potassium stimulation.     

Spinal GABA(A) receptors do not mediate the sympathetic baroreceptor reflex in the rat

Activation of baroreceptors causes efferent sympathetic nerve activity (SNA) to fall. Two mechanisms could account for this sympathoinhibition: disfacilitation of sympathetic preganglionic neurons (SPN) and/or direct inhibition of SPN. The roles that spinal GABA and glycine receptors play in the baroreceptor reflex were examined in anesthetized, paralyzed, and artificially ventilated rats. Spinal GABA(A) receptors were blocked by an intrathecal injection of bicuculline methiodide, whereas glycine receptors were blocked with strychnine. Baroreceptors were activated by stimulation of the aortic depressor nerve (ADN), and a somatosympathetic reflex was used as control. After an intrathecal injection of vehicle, there was no effect on any measured variable or evoked reflex. In contrast, bicuculline caused a dose-dependent increase in arterial pressure, SNA, phrenic nerve discharge, and it significantly facilitated the somatosympathetic reflex. However, bicuculline did not attenuate either the depressor response or sympathoinhibition evoked after ADN stimulation. Similarly, strychnine did not affect the baroreceptor-induced depressor response. Thus GABA(A) and glycine receptors in the spinal cord have no significant role in baroreceptor-mediated sympathoinhibition.