Beta-adrenoceptors in the rat parotid gland enlarged by salivariectomy and bulk diet. Effects of denervation

The numbers of beta-adrenergic receptors and level of cyclic AMP (cAMP) of the parotid gland of adult female rats were determined 4 weeks after introduction of a regimen that induced a 2-fold increase in gland weight. This regimen consisted of ablation of the submandibular-sublingual glands and substitution of the normal chow diet with a bulk diet consisting of 50% inert cellulose and 50% ground solid chow. There was a 2.4-fold increase in number (density) of beta-adrenoceptors in the enlarged parotid gland when comparison was made with parotid glands of control rats. The beta-adrenoceptor present in the enlarged and normal glands was of the beta 1 subtype. Removal of either autonomic pathway at the time of partial salivariectomy and dietary substitution was followed by a small reduction in number of beta-adrenoceptors (4-9% with either denervation), but when both nerves were removed the reduction was 25%; in magnitude, these changes were generally similar to those observed with denervated parotid glands of chow-fed rats. The norepinephrine concentration of the enlarged gland was much less than that of normal glands (reduced 38%); sympathectomy of normal or enlarged parotid glands resulted in a marked lowering of norepinephrine concentrations (to 1-5% of control levels); parasympathectomy had no effect on norepinephrine concentration of enlarged parotid glands but caused a decrease in that of the parotid of normal size. Apparently, the number of beta-adrenoceptors depends on the degree of activity of both the parasympathetic and sympathetic nerves to the parotid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nerve growth factor effects on inhibition by submandibular gland ablation or autonomic denervation of activity-induced parotid growth.

Present data confirm our earlier report that an increase in [3H]thymidine incorporation into DNA of parotid gland occurs with reintroduction of solid chow to rats previously maintained on liquid diet; if, however, the submandibular-sublingual glands are removed prior to the dietary substitution, the increase is prevented (present data and [19]). In addition, present data show that administration of nerve growth factor (NGF) to partially sialoadenectomized rats during the 2-day period of dietary change from liquid to solid diet restores thymidine values to the high levels observed following the dietary change in intact animals. However, the increase in gland size that accompanied the change in dietary consistency was not prevented by prior submandibular gland ablation, and administration of NGF had no influence on gland size or cell size. The removal of both the sympathetic and parasympathetic nerves to the parotid gland prior to the dietary manipulation also suppressed [3H]thymidine incorporation into parotid, and values did not differ from chow controls. The data show that NGF, a submandibular growth factor, has a prominent role in regulation of the autonomically-mediated hyperplastic response. However, neither the submandibular gland nor NGF has an important role in regulation of the increase in gland size that also accompanies the dietary change.     

Effects of nerve growth factor on cyclocytidine-induced growth responses of sympathectomized parotid or parotid of partially desalivated rat.

Cyclocytidine (CC), a potent antitumor agent, caused a 2.4- to 3.9-fold increase in [3H]thymidine uptake of rat parotid gland after 3 days of daily administration of the CC in a dose of 500 mg/kg body weight. Gland weight also was increased. Ablation of the submandibular-sublingual glands prior to initiation of the CC regimen prevented the usual CC-induced increase in [3H]thymidine uptake but this inhibition was partially reversed when nerve growth factor (NGF) was administered with the CC; values for [3H]thymidine uptake into parotid DNA were 81, 54, and 73% of those of glands of intact CC-treated rats. Submandibular gland ablation did not prevent the usual CC-induced increase in parotid size, and administration of NGF had no effect. Sympathectomy of the salivary glands also inhibited the thymidine increase in parotid gland usually induced by CC but in addition it also inhibited the usual CC-induced increase in gland weight. NGF, however, failed to reverse the effects of sympathectomy on [3H]thymidine uptake or gland size: both remained at the same level observed in sympathectomized parotid not given NGF.     

Studies of muscarinic receptor subtypes in salivary gland function in anaesthetized rats

The in vivo study aimed to examine whether muscarinic receptor subtypes other than muscarinic M3 receptors exert exocrine functional roles in the rat salivary glands. The effects of pirenzepine, methoctramine and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) were examined on secretion from the major salivary glands evoked by acetylcholine (0.001-10 micromol kg(-1) i.v.) in pentobarbitone-anaesthetized rats. Observations were occasionally made on glandular blood flow. 4-DAMP (0.1-100 nmol kg(-1) i.v.) markedly and equipotently inhibited the acetylcholine-evoked fluid responses in all glands. Pirenzepine (0.1 micromol kg(-1) i.v.-10 mmol kg(-1) i.v.) showed significantly lower inhibitory potency than 4-DAMP, most conspicuously in the parotid, while methoctramine (0.1 micromol kg(-1) i.v.-10 mmol kg(-1) i.v.) exerted an even lesser inhibitory effect. Also against acetylcholine-evoked blood flow increases, 4-DAMP showed a conspicuous potency. At 1 and 10 micromol kg(-1) i.v. of pirenzepine, the antagonist reduced the protein concentration in the submandibular saliva, but not in the parotid saliva. While 4-DAMP (1 and 10 nmol kg(-1) i.v.) significantly inhibited acetylcholine-evoked protein secretory responses in the submandibular glands, methoctramine (below 10 micromol kg(-1) i.v.) affected the responses in neither gland. The reduction of the protein concentration in submandibular saliva caused by 4-DAMP and pirenzepine was inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME; 30 mg kg(-1) i.p.), while L-NAME had no or only minute effects on the parotid protein secretion. Thus, in addition to muscarinic M3 receptors, other muscarinic receptors contribute to in vivo functional responses in rat submandibular and sublingual glands. While these other receptors are muscarinic M1 receptors in the sublingual gland, they may be a different subtype, possibly muscarinic M5 receptors, in the submandibular gland. However, muscarinic M1 receptors may induce indirect effects via nitric oxide in the submandibular gland.     

Regulation of salivary gland function by autonomic nerves

Oral homeostasis is dependent upon saliva and its content of proteins. Reflex salivary flow occurs at a low 'resting' rate and for short periods of the day more intense taste or chewing stimuli evoke up to ten fold increases in salivation. The secretion of salivary fluid and proteins is controlled by autonomic nerves. All salivary glands are supplied by cholinergic parasympathetic nerves which release acetylcholine that binds to M3 and (to a lesser extent) M1 muscarinic receptors, evoking the secretion of saliva by acinar cells in the endpieces of the salivary gland ductal tree. Most salivary glands also receive a variable innervation from sympathetic nerves which released noradrenaline from which tends to evoke greater release of stored proteins, mostly from acinar cells but also ductal cells. There is some 'cross-talk' between the calcium and cyclic AMP intracellular pathways coupling autonomic stimulation to secretion and salivary protein secretion is augmented during combined stimulation. Other non-adrenergic, non-cholinergic neuropeptides released from autonomic nerves evoke salivary gland secretion and parasympathetically derived vasointestinal peptide, acting through endothelial cell derived nitric oxide, plays a role in the reflex vasodilatation that accompanies secretion. Neuronal type, calcium-activated, soluble nitric oxide within salivary cells appears to play a role in mediating salivary protein secretion in response to autonomimetics. Fluid secretion by salivary glands involves aquaporin 5 and the extent to which the expression of aquaporin 5 on apical acinar cell membranes is upregulated by cholinomimetics remains uncertain. Extended periods of autonomic denervation, liquid diet feeding (reduced reflex stimulation) or duct ligation cause salivary gland atrophy. The latter two are reversible, demonstrating that glands can regenerate provided that the autonomic innervation remains intact. The mechanisms by which nerves integrate with salivary cells during regeneration or during salivary gland development remain to be elucidated.     

Beta-adrenergic receptors and cAMP levels of rat parotid and submandibular glands during chronic isoproterenol treatment

The number of cell surface beta-adrenergic receptors and the level of cyclic AMP of the parotid and the submandibular gland were examined in rats treated for up to 10 days with twice daily injections of the beta-adrenergic agonist, isoproterenol. Receptor densities of 125 +/- 8.7 fmol/mg membrane protein for the parotid and 60.1 +/- 5.6 fmol/mg for the submandibular glands were found with [3H]dihydroalprenolol (beta-adrenergic receptor antagonist) binding of glands from control rats. No change from levels of controls was found in the number of beta-receptors of the submandibular gland with chronic isoproterenol stimulation; the parotid glands, on the other hand, showed a 22% decrease in dihydroalprenolol binding from the 4th until the 8th day of treatment. By day 10 of isoproterenol treatment the parotid gland demonstrated a shift from a population consisting of primarily beta-adrenergic receptors to one consisting of equal numbers of beta 1- and beta 2-adrenoceptors. The basal level of cAMP present in cell lysates remained unchanged in the isoproterenol-treated submandibular gland while the parotid gland showed a 30-40% decrease. Control and isoproterenol-treated animals demonstrated the same time course of cAMP accumulation after a single challenge with isoproterenol.     

Identification and characterization of muscarinic cholinergic receptors in the human urinary bladder and parotid gland

The binding characteristics of [3H]quinuclidinyl benzilate (QNB) to muscarinic sites in isolated plasma membrane fractions of the human urinary bladder and parotid gland were studied. QNB binding to both preparations was of high affinity and low capacity. Mean values for the apparent dissociation constants (Kd) for binding to membrane preparations from the urinary bladder and parotid glands were 22 and 34 pM and the Bmax values 234 and 456 fmol/mg protein, respectively. Significance of difference between Kd and Bmax values from the two tissues was at the level of P less than 0.005 and P less than 0.05, respectively. QNB binding was inhibited by muscarinic receptor antagonists with varying degree of effectiveness. The mean values for the inhibition constant (Ki) were significantly lower for oxybutynin, amitriptyline, and pirenzepine but higher for secoverine in preparations of the urinary bladder than of the parotid gland. The mean Ki values for quinidine and verapamil were lower in the urinary bladder than that in the parotid gland. Carbachol exhibited a marked selectivity for the urinary bladder (about 30-fold) compared with the parotid gland. The present data obtained in two human tissues that are highly cholinergic in their innervation give support to the argument for heterogeneity of the muscarinic cholinergic receptors.     

Effects of extracorporeal shock-wave lithotripsy directed at the parotid gland on oxidative stress parameters and some trace element levels in facial nerve of rats

Introduction: This study was designed to assess the effect of extracorporeal shock‐wave lithotripsy (ESWL) exposure of the parotid gland on oxidative stress and some trace element levels in the facial nerves of rats. Methods: Twelve male Wistar albino rats were divided into two groups, each consisting of 6 animals. The rats in the first group served as controls. The left parotid glands of animals in the second group were treated with 1000 18‐kV shock waves while anesthetized with ketamine. The animals in both groups were euthanized 72 h after the ESWL treatment, and the right facial nerve was harvested for determination of oxidant/antioxidant status and trace element levels. Results: Lipid peroxidation product malondialdehyde (MDA) and antioxidant glutathione (GSH) levels increased, and the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH‐Px), decreased in the facial nerves of ESWL‐treated rats. The levels of iron, lead, manganese, and cobalt increased, and magnesium, cadmium, and copper levels decreased. Conclusions: ESWL treatment of the parotid gland may increase lipid peroxidation and decrease antioxidant enzyme activity in adjacent tissues such as the facial nerve. It also causes a decrease or increase in many mineral levels of the facial nerve, which is an undesirable condition for normal physiological function. Muscle Nerve, 2012     

Effects of electrical stimulation of autonomic nervous system on degranulation of von Ebner's gland acini

In a series of studies to understand interactions between taste sensation and salivary gland function, we are pursuing experiments to determine the autonomic nervous system control of von Ebner's lingual salivary glands. Electrical stimulation of the glossopharyngeal nerve, which contains the parasympathetic nerve supply to von Ebner's glands, caused a reduction in secretory granules of the glands in the rat. This depletion of granules could be blocked by prior administration of the parasympathetic antagonist, atropine. In contrast, electrical stimulation of the sympathetic nerve supply was ineffective in causing granule depletion in von Ebner's gland, but produced almost total degranulation in the parotid gland of the same animals. It is concluded that parasympathetic nerves exert the principal control over von Ebner's gland, acinar degranulation in the rat; this is compared with autonomic control of other salivary glands that have a dual peripheral control by parasympathetic and sympathetic innervation.     

Is the muscarinic receptor that mediates potentiation of dopamine release negatively coupled to the cyclic GMP system?

Dopamine (DA) terminals in rat corpus striatum and frontal cortex possess muscarinic receptors that mediate enhancement of the depolarization-evoked release of the catecholamine. The effects of the membrane-permeating cyclic guanosine monophosphate (cyclic GMP) analog 8-Br-cyclic GMP and of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) on the muscarinic-induced increase of DA release were investigated in striatal synaptosomes prelabeled with [3H]DA and exposed in superfusion to 15 mM KCl and to acetylcholine (ACh). Preincubation of synaptosomes with 8-Br-cyclic GMP (10-200 microM) or with IBMX (200 microM) prevented the ACh-induced enhancement of [3H]DA release, without affecting the K+-evoked release of the [3H]amine. No significant decrease of the ACh effect was observed when 8-Br-cyclic GMP or IBMX were added concomitantly with ACh to the superfusion medium. The data suggest that stimulation of presynaptic muscarinic receptors on DA terminals may produce enhancement of 3H DA release through a decrease of the intraterminal cyclic GMP content.     

Salivary gland enlargement and elevated serum amylase in bulimia nervosa.

BACKGROUND: Clinical reports have described salivary gland enlargement in bulimia nervosa, particularly in patients with elevated serum amylase concentration. The goal of the current study was to provide a controlled comparison of salivary gland size in patients with bulimia nervosa and healthy volunteers. METHODS: Subjects included 17 women with bulimia nervosa and 21 healthy female control subjects. Dimensions of the parotid and submandibular salivary glands were estimated by ultrasonography. Blood samples for amylase measurement were obtained after overnight fast. RESULTS: Parotid gland size was enlarged 36% in patients with bulimia nervosa in comparison to control subjects (p < .01). For the patient group, salivary gland size was significantly correlated with frequency of bulimic symptoms and with serum amylase concentration. CONCLUSIONS: These results provide new quantitative data demonstrating increased salivary gland size in bulimia nervosa. Further studies are needed to evaluate factors responsible for salivary gland enlargement and hyperamylasemia in this disorder.     

Activation of trigeminal nociceptive neurons by parotid PAR-2 activation in rats

To clarify involvement of protease-activated receptor-2 (PAR-2) in parotid pain, we examined whether PAR-2 activation in the parotid gland could activate trigeminal nociceptive neurons in anesthetized rats, by analyzing immunoreactive Fos as a nociceptive marker. Either the PAR-2 agonist SLIGRL-NH2 or capsaicin, injected into the parotid duct, caused expression of Fos in the trigeminal subnucleus caudalis, although the PAR-2-inactive reversed peptide had no such effect. The Fos expression caused by PAR-2 activation was inhibited by ablation of capsaicin-sensitive sensory neurons. Intraductal SLIGRL-NH2 did not increase vascular permeability in the parotid gland. Our data thus reveal that activation of PAR-2 in the parotid gland can cause activation of trigeminal nociceptive neurons via capsaicin-sensitive sensory nerves most probably by a non-inflammatory mechanism.     

Inhibitory effects of atropine and adrenergic antagonists on the changes in autonomic receptors and cyclic nucleotides of rat parotid and submandibular glands caused by sympathetic nerve stimulation

The changes in cyclic AMP (cAMP) concentration and density of beta-adrenoceptors caused by electrical stimulation of the sympathetic innervation to parotid and submandibular glands of rat did not occur when the alpha- and beta-adrenergic antagonists, phentolamine, and propranolol were administered 20 min prior to initiation of stimulation. They also did not occur when phentolamine, the beta-adrenergic antagonist, was administered alone prior to nerve stimulation, indicating that beta-adrenoceptors mediate these effects. Simultaneous administration of the alpha- and beta-antagonists also prevented the changes in densities of muscarinic receptors and cGMP concentrations usually induced by sympathetic nerve stimulation. Also, the changes in muscarinic receptors and cGMP did not occur when atropine was administered prior to nerve stimulation, nor did they occur with simultaneous administration of atropine, phentolamine + propranolol; with phentolamine alone, or propranolol alone, the effects were blocked to a large extent. Secretion was inhibited completely when both adrenergic antagonists were present during nerve stimulation, but flow rate was unchanged when atropine was present. The changes in both beta-adrenoceptors and muscarinic receptors reflect a desensitization caused by prolonged exposure to neurotransmitters released when the sympathetic nerve is stimulated. The changes are prevented when either atropine or adrenergic antagonists are present during nerve stimulation.     

The bimodal effect of cholinergic blocking agents on the human parotid gland deprived of parasympathetic control

Summary Intense salivatory reactions to atropine appear only at the second and are completely at the third stage of the postdenervational syndrome. Muscarinic receptors, emerging at these stages of denervation and causing paradoxical reactions to cholinolytics, differ from those which emerge at the first stage of denervation and from those blocked with atropine. A classical antagonist, atropine is able to discriminate between heterogenous subpopulations of these receptors, emerging at various stages of denervation. The denervated parotid gland presents, an evolving system in terms of muscarinic receptors. Atropine discriminates between the highest levels in the development of those structures and the lowest ones, since for the former it plays the role of an antagonist, and for the latter the role of an agonist.     

Effects of diet and obesity on brain α1- and α2-noradrenergic receptors in the rat

The chronic feeding of a sweetened condensed milk/corn oil diet (CM diet) to adult male rats produced significant increases in body weight and levels of plasma insulin in 34% of the rats fed this diet with respect to chow-fed controls. Levels of alpha 1-noradrenergic receptor binding were lower (32%) in the hypothalamic ventromedial nucleus (VMN) of only those rats which became obese (DIO rats) with respect to both chow-fed controls and those rats which resisted the development of obesity on the CM diet (DR rats). Also, alpha 1-noradrenergic binding was inversely proportional to body weight gain in the VMN (r = -0.831). alpha 2-Noradrenergic receptors were 30-37% lower in both the DIO and DR rats in the dorsomedial nucleus and dorsal area of the hypothalamus, and the medial dorsal area and nucleus reuniens of the thalamus. The similar decreases in alpha 2-noradrenergic receptors in both the DIO and DR rats in these areas suggested that dietary factors alone were responsible for these changes. There were no significant differences from chow-fed rats for hypothalamic dopamine (D2) or beta-noradrenergic (beta 1- and beta 2-) receptors in either DR or DIO rats. These results indicate that VMN alpha 1-noradrenergic receptors co-vary with body weight and implicate a role for alpha 1-receptors in the VMN in the central neuronal regulation of body weight.     

The effect of parasympathetic postganglionic denervation on parotid salivary protein secretion in anaesthetized sheep

Effects of unilateral parasympathetic denervation of ovine parotid glands were examined in anaesthetized sheep 21-28 days after nerve section. Parasympathetic denervation reduced the mass of the ipsilateral gland while increasing that of the contralateral gland to the extent that total gland mass was greater than in sheep with normally innervated glands. The spontaneous secretion (8.8 +/- 1.1 microl min(-1) g gland(-1)) was significantly less from denervated than from innervated glands of normal control animals (26.0 +/- 2.7 microl min(-1) g gland(-1); P< 0.01) and contained more protein. Rates of flow, and the outputs of sodium and potassium, in response to sympathetic stimulation, were similar from normally innervated and chronically denervated glands, when allowance was made for the discrepancy in weights, whereas the output of protein was significantly enhanced following parasympathetic denervation (innervated--31.4 +/- 7.3 microg g gland(-1), denervated--83.4 +/- 26.6 microg g gland(-1); P< 0.05). Intra-arterial infusions of acetylcholine (130 pmol min(-1) kg(-1)) elicited a flow of parotid saliva, the protein content of which was significantly enhanced by prior parasympathetic denervation. Intra-arterial infusions of vasoactive intestinal peptide (VIP; 2.5 pmol min(-1) kg(-1)) produced a small but statistically significant (P< 0.05) increase in the flow of parotid saliva from the contralateral, innervated but not from denervated glands. It also caused a small increase in protein output, which was significantly enhanced by prior denervation. VIP had no synergistic effect on the parotid responses to acetylcholine. The results show that the parasympathetic innervation to the parotid gland of the sheep exerts important trophic effects on the gland. Interaction of adrenergic and cholinergic receptors makes an important contribution to stimulation of the secretion of protein and prior denervation potentiates the protein responses to both acetylcholine and VIP.     

Differential activation of nitric oxide synthase through muscarinic acetylcholine receptors in rat salivary glands

Muscarinic receptors play an important role in secretory and vasodilator responses in rat salivary glands. Nitric oxide synthase (NOS) appears to be one of the multiple effectors coupled to muscarinic receptors in both submandibular and sublingual glands although some differences have been found depending on the gland studied. First, submandibular glands had a lower basal activity of nitric oxide synthase than sublingual glands and the concentration-response curve for carbachol was bell-shaped in the former but not in sublingual glands. Second, cGMP levels displayed a similar profile to that observed for NOS activity in both glands. Third, protein kinase C also coupled to muscarinic receptor activation in the glands might have a regulatory effect on nitric oxide production since its activity was higher in basal conditions in submandibular than sublingual glands and it also increased in the presence of the agonist at a concentration that inhibited NOS activity in submandibular glands. The effects appear to be partly related to the expression of a minor population of M(1) receptors in submandibular glands absent in sublingual as determined in binding and signaling experiments with the muscarinic receptor antagonist pirenzepine.     

Adrenergic nerves to the rat parotid gland originating in the contralateral sympathetic chain

The parotid gland of the rat seems to receive some adrenergic nerves from the sympathetic chain of the opposite side. This is suggested by the following evidence: after unilateral removal of the superior cervical ganglion, parotid tissue from the contralateral gland shows degeneration secretion of amylase in vitro similar to, but much smaller than that known to occur ipsilaterally. When parotid secretion is evoked parasympathetically in the anesthetized rat, superimposed stimulation of the contralateral cervical sympathetic trunk can be shown to increase the secretion of amylase into this parasympathetic saliva; as it does, much more, ipsilaterally. It may also cause an evanescent decrease of the salivary flow, suggesting that not only secretory, but also vasoconstrictor nerves had been activated. After removal of one sympathetic ganglion, some undergenerated adrenergic nerves remain ipsilaterally, as earlier demonstrated; but no such fibers can be detected when the ganglion has been removed on both sides.     

Autonomic control of protein production by the parotid gland of the sheep

The role of adrenoceptors in the control of parotid salivary function has been investigated in anaesthetized sheep. The enhancement of parotid protein output that occurs when the parasympathetic and sympathetic innervations to the gland are stimulated simultaneously in bursts at a low frequency (20 Hz for 1 s at 10-s intervals) was effectively abolished by pretreatment with propranolol (> or = 1.0 mg kg(-1), i.v., P < 0.001), without a comparable reduction in the flow of saliva or in the output of sodium or potassium. Secretion of protein was similarly augmented by simultaneous stimulation of the sympathetic innervation and an intracarotid infusion of acetylcholine (0.4-0.6 microg min(-1) g gland(-1)). This effect was also abolished by pretreatment with propranolol. Pretreatment with phentolamine (>1.0 mg kg(-1), i.v.) had no effect on the output of protein that occurred during combined stimulation of the parasympathetic and sympathetic innervations but increased the flow of saliva and the output of electrolytes. Stimulation of the parasympathetic innervation to the parotid gland caused a substantial fall in vascular resistance, which was reduced by the administration of atropine (0.5 mg kg(-1)). Stimulation of the sympathetic innervation caused a substantial rise in parotid vascular resistance in atropinized sheep. This effect was greater during continuous stimulation than during intermittent stimulation and enhanced by pretreatment with propranolol. It was virtually eliminated by pretreatment with phentolamine. It is concluded that the enhancement of protein output from the ovine parotid gland, that occurs during combined stimulation of the parasympathetic and sympathetic innervations at relatively low frequencies, depends upon interaction between cholinergic muscarinic and beta-adrenergic receptors. The vasoconstriction that occurs during sympathetic stimulation alone can be accounted for by activation of alpha-adrenoceptors.     

Plasma cyclic AMP and cyclic GMP in childhood-onset psychoses

Plasma cyclic AMP is a “second messenger” that may reflect levels of activity of important neurotransmitter receptors. Plasma cyclic AMP was measured in 18 patients with childhood autism, 7 patients with pervasive developmental disorder, and 12 age- and sex-matched healthy controls. Plasma cyclic AMP was significantly elevated by over 100% in both groups of patients with childhood-onset psychoses compared with controls. Plasma cyclic GMP, a nucleotide linked to different receptors, was not elevated, suggesting that the finding may be specific.