Intrathecal capsaicin depletes substance P in the rat spinal cord and produces prolonged thermal analgesia.

A single intrathecal injection of capsaicin depletes substance P from primary sensory neurons and causes a prolonged increase in the thermal and chemical pain thresholds of the rat but no apparent change in responses to noxious mechanical stimuli.

Avian pancreatic polypeptide phase shifts hamster circadian rhythms when microinjected into the suprachiasmatic region.

The suprachiasmatic nucleus has been identified tentatively as a circadian pacemaker. To examine the functional role of peptides found within suprachiasmatic neurons, avian pancreatic polypeptide and vasopressin were microinjected into the suprachiasmatic region. Avian pancreatic polypeptide, but not vasopressin, shifted the phase of the wheelrunning rhythm as a function of the time of its injection within the circadian cycle. Avian pancreatic polypeptide or a similar peptide may be one component of the neurochemical processes underlying entrainment to the light-dark cycle.

Elevation of plasma neurotensinlike immunoreactivity after a meal. Characterization of the elevated components.

The detection of an elevation in neurotensinlike immunoreactivity in peripheral plasma for several hours after a meal has been confirmed and shown to be primarily due to the presence of aminoterminal fragments of neurotensin (NT) rather than to NT itself. We have developed a procedure to separate and characterize these N-terminal cross-reacting substances, and to estimate the contributions of these constitutents to plasma neurotensinlike immunoreactivity. Gel chromatography of pooled plasma extracts on Sephadex G-25 followed by reverse-phase high pressure liquid chromatography indicated that peptides coeluting with NT and its N-terminal partial sequences NT(1-8) and NT(1-11) were present in plasma. Comparison of plasmas collected before and 1 h after a defined meal, in five experiments, demonstrated no change in C-terminal immunoreactivity and an 8- to 10-fold rise in N-terminal immunoreactivity. Chromatographic analysis of pooled pre- and postmeal plasma in four experiments showed that essentially all of this elevation in neurotensinlike immunoreactivity measured with an N-terminal directed antiserum was due to increases in NT(1-8) and NT(1-11), while NT itself, measured using a C-terminal directed antiserum, did not increase appreciably in peripheral plasma 1 h after the meal. Generation of tritiated substances with the same elution times as NT(1-8) and NT(1-11) did occur after incubation of [(3)H]NT with whole blood in vitro, providing supporting evidence that these fragments are metabolites of NT. The marked elevation in the circulating levels of these fragments reflects that an increased secretion of NT occurred in response to the test meal. The secreted NT may have acted as a hormone before it was metabolized, or it may only have had a local (paracrine) effect.

A neurokinin 1 receptor antagonist decreases adhesion reformation after laparoscopic lysis of adhesions in a rat model of adhesion formation.

BACKGROUND: Up to 94% of patients experience fibrous adhesions after abdominal surgery, and a significant number of these patients require a second operation for open or laparoscopic lysis of adhesions (LOA). The authors have previously shown that inhibition of the binding of tachykinin ligands to the neurokinin 1 receptor (NK-1R) using the neurokinin 1 receptor antagonist (NK-1RA) CJ-12,255 decreases primary adhesion formation and upregulates the peritoneal fibrinolytic system in a rat model. Whereas most studies have focused on the prevention of primary adhesions, few have addressed adhesion reformation after LOA. This study aimed to determine the effects of NK-1RA administration on adhesion reformation and peritoneal fibrinolytic activity after laparoscopic LOA. METHODS: Adhesions were induced in 31 rats using our previously described ischemic button model. The rats underwent laparoscopy 7 days later, during which adhesions were scored and lysed followed by administration of the NK-1RA or saline. Then 7 days after LOA, 23 rats were killed and adhesions were scored. Eight rats also were killed 24 h after the LOA to obtain peritoneal tissue and fluid, which were analyzed for tissue plasminogen activator (tPA) mRNA expression and peritoneal fibrinolytic activity by reverse transcriptase-polymerase chain reaction (RT-PCR) and bioassay, respectively. RESULTS: At laparoscopy, 79% +/- 3% of the buttons formed adhesions. In the saline-administered control animals, 42% +/- 3.2% of the buttons reformed adhesions after LOA (p < 0.05), whereas in the animals that received the NK-1RA, 18.2% +/- 3.5% of the buttons reformed adhesions (p < 0.05). As compared with control animals, NK-1RA administration increased tPA mRNA levels by 38% and fibrinolytic activity sixfold (p < 0.05; 7.0 +/- 2.1 U/ml vs 1.2 +/- 0.54 U/ml). CONCLUSIONS: When administered during laparoscopic LOA, an NK-1RA significantly upregulates peritoneal fibrinolytic activity and decreases adhesion reformation.

Diminished flare response in neuropathic diabetic patients. Comparison of effects of substance P, histamine, and capsaicin.

The flare response in skin largely depends on an intact primary sensory fiber, the C-fiber. We measured the flare response to the intradermal injection of substance P, histamine, and capsaicin in control subjects and in diabetic patients with and without clinically obvious polyneuropathy. The neuropathic diabetic patients had a reduced flare response to substance P, histamine, and capsaicin, compared with control and nonneuropathic diabetic subjects. The smaller flare response in the neuropathic diabetics after capsaicin administration suggested a dysfunction of the peripheral component of the C-fiber. Alternatively, dysfunction of the mast cell or vascular reactivity may contribute to the diminished flare. Because C-fibers participate in nociception in addition to the flare response, the findings of this study, by a method that permits a quantifiable measurement of the function of peripheral sensory neurons in diabetic subjects, has potential usefulness in evaluating sensory neuropathy in diabetic patients.

Substance P stimulates luteinizing hormone secretion from anterior pituitary cells in culture.

Substance P (SP) is present in the anterior pituitary gland (AP), and its concentration there is regulated by the hormonal status of the animal. The observation that SP is releasable from hemipituitaries in a K(+)-stimulated, Ca(2+)-dependent manner and the demonstration of SP-binding sites in the AP have led to the suggestion that SP participates in a paracrine or autocrine manner in the regulation of AP function. Contradictory reports of the effects of SP on the secretion of AP hormones, particularly LH, led us to address the question of whether SP can act directly on the AP to effect LH secretion. We found that SP (100 nM) can stimulate LH release (300-400% of control values) in short term cultures of AP cells and that this effect varies as a function of the age and sex of the animal. There was no significant effect of SP on the release of LH from AP cells of male and female prepubertal rats (20-30 days). During the peripubertal period (30-35 days), a sharp increase in the response to SP occurred in both sexes. This responsiveness was dose dependent and persisted at all ages studied in AP cells from the female rat. In contrast, the responsiveness of AP cells from male rats that developed during the peripubertal period diminished during maturation and was absent after 60 days of age. When adult female rats were exposed to androgens for 6 weeks in vivo and tested for the ability of SP to stimulate the LH secretion, the response was significantly diminished. These studies support the speculation that SP has a functional role in the secretion of LH.

Neurotensin in plasma: immunochemical and chromatographic character of acid/acetone-soluble material.

Using a RIA, we have examined the character of the neurotensin-like (NT-like) peptides present in acid/acetone extracts of animal and human plasma. Plasma immunoreactivity was measured using four antisera with different specificities. Antisera which were directed against the COOH-terminus of NT measured higher levels of plasma immunoreactivity than did antisera with NH2-terminal or mixed specificity. Chromatographic fractionation of bovine plasma extracts revealed the presence of multiple substances, one of which was chromatographically and immunochemically indistinguishable from NT. We estimate the plasma level of this component to be about 15-25 fmol/ml, which is 30-50% of the measurement obtained on unfractionated extracts using antiserum HC-8. Several other components were also identified which behaved as though they were smaller than NT and seemed to share with NT four to eight of its COOH-terminal amono acids. One eluted from Sephadex G-25 in the region of the NT-like peptide previously identified in extracts of gastric mucosa. Infusion of synthetic NT into rats for 30 min did not result in the formation of these COOH-terminal relatives of NT. Our results argue strongly for the presence of NT in plasma and also indicate that other peptides, sharing COOH-terminal homologies with NT, appear in plasma, possibly from the stomach, liver, and other as yet unidentified source(s).

Thyroid hormone status regulates preprotachykinin-A gene expression in male rat anterior pituitary.

Substance P (SP) and substance K (SK) are mammalian tachykinin peptides derived from a single preprotachykinin-A (PPT-A) gene and are widely but selectively distributed in neural and endocrine tissues. SP is present in the rat anterior pituitary, and its content there varies with the thyroid status of the animal. The present studies were undertaken to determine whether the PPT-A gene is expressed in the anterior pituitary and if so, whether PPT-A messenger RNA (mRNA) abundance is regulated by thyroid hormone status. Male rats were surgically or chemically thyroidectomized or made hyperthyroid by thyroid hormone (T3) injection. Total RNA was isolated from individual anterior pituitaries, and PPT-A mRNA abundance was determined by dot blot procedures. In parallel groups of rats, anterior pituitaries were extracted for measurement of SP and SK by specific RIAs. Surgical or chemical thyroidectomy increased PPT-A mRNA abundance 4 to 6-fold and increased both SP and SK content in the anterior pituitary. Administration of T3 to thyroidectomized rats reversed the increase in both PPT-A mRNA abundance and SP and SK content in the adenohypophysis. T3 administration to euthyroid rats also decreased PPT-A mRNA abundance and SP and SK content in the anterior pituitary. The coordinate presence of PPT-A mRNA with SP and SK in the anterior pituitary strongly suggests that these peptides are synthesized within this gland.

Hyperglycemic effect of neurotensin, a hypothalamic peptide.

A hypotensive, gut-contracting peptide neurotensin (NT), recently isolated from bovine hypothalami, has been found to produce hyperglycemia within minutes after iv injection into anesthetized rats. The dose-response relationship (deltaglucose, 15 min after injection) was linear over the range 30-200 pmol/100 g BW. NT did not alter the disappearance rate of [14C]glucose from plasma during the development of the hyperglycemia. However, the peptide caused a fall in liver glycogen (52 +/- 6.5 to 41 +/- 3.3 mg/g) and a 7-fold increase in the activity of the 5'-AMP independent form of liver glycogen phosphorylase. Activation of liver glycogen phosphorylase did not occur in vitro under conditions found suitable for demonstrating the effectiveness of glucagon, suggesting the possible involvement of an intermediary substanc(s) in vivo. Acute adrenalectomy did not prevent the response. Hypophysectomized rats (4 days post-operative) were less sensitive to NT, perhaps as a consequence of their diminished liver glycogen levels (normal, 52 +/- 6.5 mg/g; hypophysectomized, 23 +/- 1.8 mg/g); however, the presence of the pituitary was not essential for this response. NT was also effective in rats with hereditary diabetes insipidus (Brattleboro strain). At the time intervals sampled, radioimmunoassayable plasma levels of growth hormone, glucagon, and insulin were not significantly changed after injection of NT into normal rats. Pretreatment of rats with reserpine (7 mg/kg), morphine sulfate (10 mg/kg), propranolol (5mg/kg), or phenoxybenzamine (10 mg/kg) did not prevent the response. These findings characterize the action of NT on liver glycogen metabolism and blood glucose levels, but a physiological role for NT in this regard remains to be demonstrated.

The differential effects of thyroid and gonadal hormones on substance P content in the anterior pituitary of the prepubertal rat.

The effects of thyroid and gonadal status on the content of substance P in the anterior pituitary (AP-SP) were examined in prepubertal rats. A sex difference in AP-SP is evident by age 50 days [males, 287 +/- 35 fmol/mg protein (mean +/- SE); females, 103 +/- 17; P less than 0.05], and this difference becomes greater by 75 days (males, 543 +/- 54; females, 146 +/- 11.5; P less than 0.01). Hypothyroidism was induced in male and female pups by giving lactating dams 0.1% methimazole (wt/vol) in their drinking water after parturition. There was a marked and significant increase in AP-SP in 21-day-old hypothyroid compared to euthyroid control pups. Male pups were made thyrotoxic by daily treatment with T4 (10 micrograms/rat, sc) from age 8 to 15 days. AP-SP was 4 times lower in the thyrotoxic than in the euthyroid pups (P less than 0.001). Rats ovariectomized at age 22 days and killed on day 35 revealed no change in AP-SP, in contrast to the rise in AP-SP in the ovariectomized adult rat. Female pups were treated with dihydrotestosterone (DHT; 50 micrograms/day) or testosterone (50 micrograms/day) from age 8-20 days. Neither androgen induced a change in AP-SP. Female pups which received estradiol (E2; 0.5 micrograms/day) or testosterone (75 micrograms/day) from age 8-20 days also had no change in AP-SP. As opposed to the lack of effect of E2 and DHT on AP-SP in female rats younger than 22 days, E2 (1 microgram/100 g BW daily) caused a decrease and DHT (100 micrograms/100 g BW daily) caused an increase in AP-SP in female rats treated from 22-35 days of age [E2, 91 +/- 6.9; DHT, 226 +/- 31 (P less than 0.05 vs. control for both); control, 154 +/- 13]. We conclude that the responsiveness of AP-SP to alterations in thyroid status is present at the youngest age studied. In contrast, the responsiveness of AP-SP to changes in the levels of gonadal steroids is absent in the infantile period and requires a maturational process that becomes evident during the juvenile state of sexual development.

Neurotensin neurons in the rat hypothalamus: an immunocytochemical study.

Neurotensin was localized in the hypothalamic tissues of adult Sprague-Dawley rats by immunoperoxidase techniques. Visualization of perikarya was greatly enhanced by intraventricular administration of colchicine. Many perikarya containing neurotensin-like immunoreactivity were seen in the medial preoptic area, the periventricular hypothalamus, the parvocellular portion of the paraventricular nucleus, the arcuate nucleus, and the lateral hypothalamus in the perifornical area. There were moderate numbers of cell bodies in the ventral portion of the anterior hypothalamus, the dorsomedial nucleus, and the posterior hypothalamus. No positive cells were seen in the suprachiasmatic, ventromedial, or mammillary nuclei. Reactive fibers were generally distributed in the same regions as cell bodies. Additional dense collections were seen in the lateral part of the zona externa of the median eminence, the pituitary stalk, the posterior mammillary nucleus, and the most lateral portions of the hypothalamus at the medial edge of the crura cerbri. There were smaller numbers of fibers found in the pre-mammillary and posterior hypothalamic nuclei and the posterior pituitary gland. These results indicate that the neurotensin system in the hypothalamus is very extensive and complex, as it is in many other brain regions. Neurons and fibers are found in many hypothalamic areas, including projections to the hypophysial portal system in the median eminence, suggesting that neurotensin may affect neuroendocrine mechanisms at several levels, including the anterior pituitary gland.

The aromatization of androgens by primary monolayer cultures of fetal rat hypothalamus.

Aromatization of [3H]androstenedione and [3H]19-hydroxyandrostenedione to [3H]estrone has been demonstrated to occur in one to two week old primary monolayer cultures of fetal rat hypothalamus. Three times more estrone is produced from 19-hydroxyandrostenedione than from androstenedione in four day incubations. Cultures treated with cytosine arabinoside have 50% less cellular protein and produce three times more estrone from either substrate than untreated cultures. Time course experiments using cultures treated with cytosine arabinoside indicate that aromatization of 19-hydroxyandrostenedione is linear for three days and can be quantitatively measured within the first two to eight hours of incubation.

Evidence that neurotensin participates in the central regulation of the preovulatory surge of luteinizing hormone in the rat.

Neurotensin (NT) has been implicated in the central regulation of LH and PRL secretion in the rat. We investigated the importance of NT release to the neural events that trigger the preovulatory LH surge and coincident PRL surge, using as our animal model ovariectomized (OVX) rats treated with estrogen and progesterone to induce reliable and robust surges. To interfere with the action of endogenous NT in the basal forebrain, we administered a NT antiserum (NTAS) in a series of bilateral microinjections aimed at the anterior border of the medial preoptic area. One week after OVX, rats bearing cerebral guide cannulae received Silastic capsules (3 x 15 mm; sc) containing 17 beta-estradiol. Two days later, beginning at 0830 h, conscious rats were administered either NTAS or control serum bilaterally in a series of four 100-nl injections spaced at 30-min intervals. After an initial blood sample, rats received progesterone (4 mg, sc) at 1200 h; blood samples were then taken at 1-h intervals from 1400-2100 h. Blood samples were obtained from conscious, freely moving rats via a chronic atrial catheter implanted previously. Plasma levels of LH and PRL were measured by RIA, and the location of microinjection sites was verified histologically. Administration of NTAS caused a 66% reduction in the magnitude of the LH surge without altering its timing, whereas the PRL surge was unaffected. These results provide strong evidence that NT in the basal forebrain participates in the steroid-induced LH surge and suggest that NT plays a role in the preovulatory LH surge.

The effects of gonadal steroids on the content of substance P in the rat anterior pituitary.

The effects of gonadectomy and of the administration of gonadal steroids on the content of substance P in the anterior pituitary (AP-SP) were studied in adult rats. The effect of gonadal status on the AP-SP content of thyroidectomized (TX) rats was also studied. We have confirmed that the AP-SP content in adult males is higher than that in adult females. Ovariectomy (OVX) caused an increase in AP-SP content which was apparent 6 days after surgery. Estradiol (E2; 2 micrograms/rat daily) administered for 13 days beginning the day after OVX prevented the increase in AP-SP content induced by OVX. Orchiectomy of adult rats had no effect on AP-SP content 14 and 45 days after surgery. E2 administered to adult female rats for 13 days caused a reduction in the AP-SP content, whereas dihydrotestosterone (0.2 mg/rat daily for 13 days) caused an increase that was even more pronounced in TX rats. E2 administration to TX adult female rats caused a significant decrease in the AP-SP content both when treatment was begun on the day after surgery or 2 weeks later. Administration of T4 (1.5 and 25 micrograms/100 g BW daily for 7 days) to rats made hypothyroid by thyroidectomy 2 weeks earlier abolished the increase in AP-SP content seen in TX animals. Neither E2 nor dihydrotestosterone had an effect on the substance P content of any of the brain regions examined. The AP-SP content of pregnant or lactating rats was not different from that of age-matched controls. The content of substance P in the AP and median eminence did not vary significantly throughout the estrous cycle. The data indicate that AP-SP content is dependent on the gonadal status of the animal and that gonadal steroids interact with thyroid hormones in the regulation of substance P turnover in the AP.

Regulation by thyroid hormone of the concentration of substance P in the rat anterior pituitary.

The content of immunoreactive substance P (iSP) in the male rat anterior pituitary was measured after thyroidectomy and excess T4 administration. Baseline values for iSP content (mean +/- SE, 400 +/- 37 fmol/mg protein) increased progressively after thyroidectomy (4 days, 893 +/- 100; 6 days, 1321 +/- 242; 14 days, 1897 +/- 509). Administration of pharmacological doses of T4 (50 micrograms daily) for 2 and 14 days significantly decreased anterior pituitary iSP content (2 days, 196 +/- 30; 14 days 138 +/- 12). Thyroid status did not affect iSP content in the hypothalamus, caudate nucleus, amygdala, or brainstem. Partial chemical characterization of SP immunoreactivity in the anterior pituitary was obtained by gel permeation chromatography on Sephadex G-25, high pressure liquid chromatography, and the use of two antisera in RIAs, one directed against the amino-terminus and one directed against the carboxyl-terminus of the peptide. SP in the anterior pituitary was readily releasable in vitro by 44 mM potassium chloride in a calcium-dependent manner. The present study demonstrates that the concentration of iSP in the rat anterior pituitary is affected by the thyroid status of the animal and supports the probability that thyroid hormones participate in the regulation of the synthesis and/or release of iSP from the anterior pituitary.

Alcohol and fatty acid stimulation of neurotensin release from rat small intestine.

We have previously reported that neurotensin (NT) is released from the small intestine and elevated in the hepatic-portal circulation in response to the perfusion of the small intestine with a micellar solution of oleic acid. In order to determine the minimum acyl chain length and whether the presence of a carboxylic acid is necessary for the stimulation of NT release, the small intestine of anesthetized rats was perfused with test solutions of fatty acids of 2-, 4-, 8-, or 18-carbons or fatty alcohols of 2-, 4-, or 8-carbons at a concentration of 1 mM prepared in 2.4 mM taurodeoxycholate in 0.9% NaCl. Blood samples, collected from the superior mesenteric vein immediately before the start of the test perfusion and at 15-min intervals thereafter, were extracted immediately and radioimmunoassayed for NT-like immunoreactivity (NTLI) with a C-terminal-directed antiserum. Perfusions of fatty acids with 4 or more carbons and alcohols of 2 or more carbons resulted in a significant elevation (P less than 0.05) in plasma levels of NTLI above the values obtained before the onset of perfusion. Perfusions with ethanol resulted in a value of 4.3 +/- 0.03 mg/dl (SEM) in blood from the superior mesenteric vein while there was no increase in ethanol levels in the peripheral circulation. Perfusion with taurodeoxycholate and 0.9% NaCl alone had no significant effect on plasma levels of the NTLI. In order to characterize the chemical nature of the elevated NTLI, plasma samples from animals perfused with test solution were collected, extracted, pooled, and subjected to HPLC. NT and its N-terminal metabolite, NT(1-8), were quantitated. NT was defined as material having the same retention time as synthetic NT standard and having comparable measurements using N- and C-terminal-directed antisera. Perfusions of fatty acids of four or more carbons and alcohols of two or more carbons resulted in a 2- to 4-fold increase of both NT and NT(1-8) levels in plasma. It is particularly interesting that perfusion with ethanol (2-carbons) causes an elevation in plasma NT, because perfusion with acetic acid (2-carbons) does not increase NTLI. The fact that perfusion of ethanol is effective in releasing intestinal NT suggests that NT may mediate some of the biological effects observed after the consumption of alcohol.

Estrogen induces neurotensin/neuromedin N messenger ribonucleic acid in a preoptic nucleus essential for the preovulatory surge of luteinizing hormone in the rat.

Ovarian steroids act on unidentified neurons to trigger preovulatory secretion of GnRH. In the rat, important steroid target cells reside in the anterior medial preoptic nucleus (AMPN), a sexually dimorphic structure essential for stimulatory effects of ovarian steroids on LH secretion. The AMPN contains neurotensin (NT)-immunoreactive neurons, and immunoneutralization of NT in the preoptic region markedly attenuates steroid-induced LH surges. Using probes derived from the rat gene that encodes NT and neuromedin N (NT/N), we investigated the ability of estrogen to influence NT/N mRNA levels in the AMPN. Ovariectomized rats were treated for 14 days with sham capsules or capsules that produce supraphysiological serum levels of 17 beta-estradiol (250 +/- 20 pg/ml). As determined by in situ hybridization, estradiol markedly altered the distribution of NT/N mRNA in the medial preoptic region, causing a striking increase in NT/N mRNA abundance specifically in the AMPN and adjacent medial preoptic nucleus (MPN). In contrast, estradiol caused no obvious changes in labeling in the lateral septum, diagonal band of Broca, bed nucleus of the stria terminalis, and lateral preoptic area. The distribution of NT/N mRNA in the AMPN of normal male rats closely resembled that in ovariectomized rats, where labeled cells were rarely observed. Microdissection and S1 nuclease protection analysis were used to quantitate the effect of estradiol on NT/N mRNA levels. Supraphysiological estradiol treatment for 14 days caused a 3.4-fold increase (P less than 0.0002) in NT/N mRNA levels in the combined AMPN/MPN, whereas levels in the central amygdaloid nucleus remained constant, providing further evidence of regional specificity. Forty-eight hours of estradiol treatment, at concentrations (60 +/- 1 pg/ml) similar to those observed on the morning of proestrus, caused a 1.8-fold increase (P less than 0.001) in NT/N mRNA levels in the AMPN/MPN, indicating that the time course of NT/N mRNA induction by estrogen is compatible with events of the normal estrous cycle. Together with previous findings, our results strongly suggest that NT neurons mediate, directly or indirectly, stimulatory effects of ovarian steroids on GnRH secretion.

Cardiovascular effects of a specific nonpeptide antagonist of substance P (NK-1) receptor in DOCA-salt hypertension.

The neurotransmitter substance P acts also as a potent vasodilator. Its participation in the pathogenesis of deoxycorticosterone acetate (DOCA)-salt hypertension was evaluated by an acute infusion of a newly synthesized, potent, specific nonpeptide antagonist of substance P at the NK-1 receptor, the agent CP 96,345. In conscious unrestrained rats, CP 96,345 induced significant and sustained increases in mean arterial pressure of DOCA-salt rats but only small, transient, and nonsignificant rises in blood pressure of sham-treated control rats. The rise in blood pressure was not accompanied by changes in heart rate. Maximal blood pressure increase in DOCA-salt rats was 31.7 +/- 14.8 mm Hg. In a second series of experiments, the hemodynamic effects of this antagonist were evaluated under anesthesia in both DOCA-salt and sham-treated control rats by the thermodilution method. During CP 96,345 infusion, sustained increases in cardiac index and stroke volume and decreases in total peripheral resistance were observed in both DOCA-salt and control rats. In DOCA-salt rats, cardiac index rose by 79.4%, while total peripheral resistance fell by 27.9% of the baseline values. In control rats, the changes were smaller (+27.2% and -22.5%, respectively). Stroke volume changed in parallel to cardiac output in both groups. The data suggest that acute blockade of NK-1 receptors increases blood pressure in DOCA-salt rats mainly by an increase in cardiac output. We conclude that endogenous substance P tends to counteract the DOCA-salt-induced elevation of blood pressure by modulating both cardiac output and peripheral resistance.

Role of substance P in blood pressure regulation in salt-dependent experimental hypertension.

The participation of substance P in the pathogenesis of five models of experimental hypertension, ie, DOCA-salt, subtotal nephrectomy, one-kidney-one clip renovascular, two-kidney-one clip renovascular, and spontaneous hypertension, was evaluated via an acute infusion of a newly synthesized potent, specific nonpeptide antagonist of substance P at the NK-1 receptor, the agent CP 96,345. In conscious unrestrained rats, CP 96,345 induced significant and sustained increases in mean arterial pressure of DOCA-salt, subtotal nephrectomy, and one-kidney-one clip renovascular hypertensive rats but only small and nonsignificant changes in blood pressure of two-kidney-one clip renovascular and spontaneously hypertensive rats. CP 96,345 had no effect on the blood pressure of sham-treated controls and Wistar-Kyoto rats. This NK-1 receptor antagonist did not significantly affect the heart rate of any experimental model studied. The data suggest that endogenous substance P may act as a partial counterregulatory mechanism against vasoconstriction in models of salt-dependent hypertension.

The common C-terminal sequences of substance P and neurokinin A contact the same region of the NK-1 receptor.

Although neurokinin A (NKA), a tachykinin peptide with sequence homology to substance P (SP), is a weak competitor of radiolabeled SP binding to the NK-1 receptor (NK-1R), more recent direct binding studies using radiolabeled NKA have demonstrated an unexpected high-affinity interaction with this receptor. To document the site of interaction between NKA and the NK-1R, we have used a photoreactive analogue of NKA containing p-benzoyl-L-phenylalanine (Bpa) substituted in position 7 of the peptide. Peptide mapping studies of the receptor photolabeled by (125)I-iodohistidyl(1)-Bpa(7)NKA have established that the site of photoinsertion is located within a segment of the receptor extending from residues 178 to 190 (VVCMIEWPEHPNR). We have previously shown that (125)I-BH-Bpa(8)SP, a photoreactive analogue of SP, covalently attaches to M(181) within this same receptor sequence. Importantly, both of these peptides ((125)I-iodohistidyl(1)-Bpa(7)NKA and (125)I-BH-Bpa(8)SP) have the photoreactive amino acid in an equivalent position within the conserved tachykinin carboxyl-terminal tail. In this report, we also show that site-directed mutagenesis of M(181) to A(181) in the NK-1R results in a complete loss of photolabeling of both peptides to this receptor site, indicating that the equivalent position of SP and NKA, when bound to the NK-1R, contact the same residue.