Calcitonin gene-related peptide (CGRP): Responsible for the increased blood flow induced by the stimulation of sensory nerves

Calcitonin gene-related peptide (CGRP) is a potent vasodilator neuropeptide contained in sensory nerves. We have examined the relative contribution of CGRP and substance P-like peptides (with which CGRP is commonly colocalized) to the increase in blood flow induced by the stimulation of sensory nerves. The sensory nerve stimulant capsaicin increased blood flow in rabbit and rat skin and this effect was substantially inhibited by the CGRP antagonist CGRP_8–37. Further, electrical stimulation of the rat saphenous nerve led to an increase in blood flow which was significantly inhibited by CGRP_8–37. CGRP_8–37 also had a partial inhibitory effect on oedema formation, an effect which is suggested to be a consequence of the inhibition of blood flow.     

The action of a calcitonin gene-related peptide antagonist in human skin

The CGRP antagonist, CGRP_8-37, antagonized the ability of CGRP to increase blood flow in human skin and cause erythema. The mechanism of the antagonist effect of CGRP_8-37 on the CGRP-induced erythema was an increase by the antagonist of the rate of decay of the CGRP-induced erythema. Since CGRP_8-37 activates rat peritoneal mast cells to release their granular contents, we conclude that the degradation of CGRP by protease released from skin mast cells by CGRP_8-37. It was not possible to demonstrate any antagonistic effect of CGRP_8-37 on the CGRP receptor mediating increased blood flow in human skin.     

CGRP (8–37) reduces the duration but not the maximal increase of antidromic vasodilation in dental pulp and lip of the rat

In this study the newly developed blockers of substance P (CP-96,345) and calcitonin gene-related peptide (CGRP_{8 37}) were used to examine whether substance P and CGRP are involved in the afferent nerve induced vasodilation in the rat lower incisor pulp and lip. Electrical stimulation of the inferior alveolar nerve (10 V, 2 ms, 10 Hz, 30 s) in the presence of phenoxybenzamine (3 mg kg¹) induced an immediate vasodilation in the pulp and lip (52 and 186% increase in blood flow respectively, n = 12) with a long duration. Infusion of 2 mg kg^-1 CP-96,345, a dose that inhibited the vasodilator effects of substance P (5–25 ng kg^-1) in oral tissues, did not have any effect on antidromic vasodilation in either tissue. After infusion of CGRP_{8 37} (0.3 mg kg^-1) the duration of the antidromic vasodilation in the pulp and lip was significantly reduced by 72 and 67% respectively (P < 0.05, n= 4), whereas the maximal increase of the response was unaffected. The blocking effect of the drug was short-lasting. When combined infusions of CP-96,345 and CGRP_{8 37} were given, a similar reduction in the duration of antidromic vasodilation in the pulp and lip occurred but in this case the amplitude of vasodilation in the pulp was reduced (from 35 + 9 to 12 + 3%, P < 0.05, n= 4). However, in the lip, the amplitude of vasodilation was not significantly reduced. The present findings indicate an involvement of CGRP in the mediation of the late phase of antidromic vasodilation in rat oral tissues and a role of substance P in the initiation of antidromic vasodilation in the incisor pulp.     

The calcitonin gene-related peptide (CGRP) antagonist CGRP8–37 blocks vasodilatation in inflamed rat skin: involvement of adrenomedullin in addition to CGRP

The neuropeptide calcitonin gene-related peptide (CGRP) is a potent microvascular vasodilator in rat skin and effects are antagonised by CGRP_8–37. In this study, CGRP_8–37 significantly (P<0.05) inhibited the time-dependent (3–5 h) increase in skin blood flow measured in the anaesthetised rat, after intradermal administration of the inflammatory cytokine interleukin-1β (3 pmol/site), indicating the involvement of CGRP1 receptors. The CGRP-related peptide adrenomedullin (ADM) is also a potent vasodilator in rat skin, with effects antagonised by CGRP_8–37. We show that ADM mRNA expression is increased in rat skin after treatment with IL-1β and that the IL-1β-induced blood flow is blocked by a selective ADM antibody (P<0.05). Thus ADM is expressed locally in the inflamed cutaneous microvasculature where it can, in addition to, or as an alternative to CGRP, contribute to IL-1β-induced vasoactive effects.     

The contribution of calcitonin gene-related peptide (CGRP) to neurogenic vasodilator responses

The neuropeptide calcitonin gene-related peptide (CGRP) is a potent vasodilator in the microcirculation of many tissues including the skin and joint. In order to elucidate the mechanism of endogenous CGRP release, we have used a multiple site¹³³Xe clearance technique to measure local blood flow changes in response to agents injected intradermally in the rabbit. Capsaicin (100 nmol/site) and human αCGRP (3 pmol/site) stimulated similar increases in blood flow and, in both cases, the effect was totally abolished by the CGRP antagonist, CGRP_8–37 (1 nmol/site). By contrast, the nitric oxide synthase inhibitorl-nitro arginine methyl ester (l-NAME, 30 nmol/site) had little effect on human αCGRP-induced vasodilation, but caused significant inhibition of the response to capsaicin (p<0.05). These results show that increased blood flow in rabbit skin caused by exogenous CGRP is independent of nitric oxide. In addition, however, they suggest that nitric oxide is required for either the release of endogenous CGRP from capsaicin-sensitive nerves or its subsequent activity.     

Involvement of capsaicin-sensitive afferent nerves in the proteinase-activated receptor 2–mediated vasodilatation in the rat dura mater

Neurogenic inflammation of the dura mater encephali has been suggested to contribute to the mechanisms of meningeal nociception and blood flow regulation. Recent findings demonstrated that the rat dura mater is innervated by trigeminal capsaicin-sensitive peptidergic nociceptive afferent nerves which mediate meningeal vascular responses through activation of the transient receptor potential vanilloid type 1 (TRPV1) receptor. The present work explored the functional significance of the capsaicin-sensitive subpopulation of dural afferent nerves via their contribution to the meningeal vascular responses evoked through activation of the proteinase-activated receptor 2 (PAR-2). The vascular responses of the dura mater were studied by laser Doppler flowmetry in a rat open cranial window preparation. Topical applications of trypsin, a PAR-2-activator, or Ser–Leu–Ile–Gly–Arg–Leu–amide (SLIGRL-NH₂), a selective PAR-2 agonist peptide, resulted in dose-dependent increases in meningeal blood flow. The SLIGRL-NH₂-induced vasodilatation was significantly reduced following capsaicin-sensitive afferent nerve defunctionalization by prior systemic capsaicin treatment and by pretreatment of the dura mater with the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP_8-37. Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) an unspecific inhibitor of nitric oxide (NO) production, but not 1-(2-trifluoromethylphenyl) imidazole (TRIM), a neuronal NO synthase inhibitor, also inhibited the vasodilator response to SLIGRL-NH₂. The vasodilator responses elicited by very low concentrations of capsaicin (10 nM) were significantly enhanced by prior application of SLIGRL-NH₂. The present findings demonstrate that activation of the PAR-2 localized on capsaicin-sensitive trigeminal nociceptive afferent nerves induces vasodilatation in the dural vascular bed by mechanisms involving NO and CGRP release. The results indicate that the PAR-2-mediated activation and sensitization of meningeal capsaicin-sensitive C-fiber nociceptors may be significantly implicated in the pathophysiology of headaches.     

Influence of methanandamide and CGRP on potassium currents in smooth muscle cells of small mesenteric arteries

Cannabinoids have potent vasodilatory actions in a variety of vascular preparations. Their mechanism of action, however, is complex. Apart from acting on vascular smooth muscle or endothelial cannabinoid receptors, several studies point to the activation of type 1 vanilloid (TRPV1) receptors on primary afferent perivascular nerves, stimulating the release of calcitonin gene-related peptide (CGRP). In the present study, the direct influence of the cannabinoid methanandamide and the neuropeptide CGRP on the membrane potassium ion (K⁺) currents of rat mesenteric myocytes was explored. Methanandamide (10 μM) decreased outward K⁺ currents, an effect similar to that observed in smooth muscle cells from the rat aorta. Conversely, CGRP (10 nM) significantly increased whole-cell K⁺ currents and this effect was abolished by preexposure to tetraethylammonium chloride (1 mM) or iberiotoxin (100 nM), inhibitors of large-conductance calcium-dependent K (BK_Ca) channels but not by glibenclamide (10 μM), an inhibitor of ATP-dependent K channels. In the presence of the CGRP receptor antagonist CGRP_8-37 (100 nM), the adenylyl cyclase inhibitor SQ22536 (100 μM), or the protein kinase A inhibitor Rp-cAMPS (10 μM), CGRP had no effect. These findings show that methanandamide does not increase membrane K⁺ currents in smooth muscle cells of small mesenteric arteries, supporting an indirect mechanism for the reported hyperpolarizing influence in this vessel. Moreover, CGRP acts directly on these smooth muscle cells by increasing BK_Ca channel activity in a CGRP receptor and cyclic adenosine monophosphate-dependent way. Collectively, these data indicate that methanandamide relaxes and hyperpolarizes intact mesenteric vessels by releasing CGRP from perivascular nerves.     

Transmural field stimulation-induced relaxation in the rat common hepatic artery.

Hepatic arteries are reportedly innervated by vasoconstrictor and vasodilator nerves. Experiments were carried out to investigate the possible involvement of calcitonin gene related peptide (CGRP) and nitric oxide as neurotransmitters during the relaxation of the rat common hepatic artery produced by transmural electrical field stimulation (ES). Common hepatic arteries were excised under ether-anesthesia from 6 weeks-old female rats, and isometric tensions recorded from endothelium-damaged ring preparations. In the presence of atropine and guanethidine, ES relaxed arteries which had been previously contracted with vasopressin. The relaxation response to ES was attenuated by either tetrodotoxin or capsaicin-pretreatment. CGRP induced a concentration-dependent relaxation, which was inhibited by the CGRP antagonist CGRP(8-37). The ES-induced relaxation was attenuated either slightly by the nitric oxide synthesis inhibitor L-nitroarginine (L-NNA) or markedly by CGRP(8-37). The relaxation response was nearly abolished in the presence of both CGRP(8-37) and L-NNA. These results may indicate that the nerve stimulation-induced vasodilatation of the rat common hepatic artery is mediated mainly by CGRP and partly by nitric oxide.     

Evidence for central timing of rhythmical mastication

1. Electrical stimulation of the vagus nerves produced a parallel increase in gastric acid secretion and gastric mucosal blood flow.

2. Gastric acid secretion and mucosal blood flow, stimulated by pentapeptide infusions or by vagal stimulation, were markedly and equally reduced by electrical stimulation of splanchnic nerve fibres.

3. The splanchnic stimulated reduction in acid and mucosal blood flow occurred only when the rise in blood pressure, normally associated with splanchnic stimulation, was prevented by inclusion of a pressure reservoir in the circulation.

4. There was evidence that the effect of the splanchnic nerves was not mediated by release of adrenaline from the adrenal medulla.

     

Activation of renal pelvic chemoreceptors in rats: role of calcitonin gene-related peptide receptors

Substance P and calcitonin gene-related peptide (CGRP) increase afferent renal nerve activity (ARNA). A substance P receptor antagonist but not a CGRP receptor antagonist, h-CGRP (8–37), blocks the ARNA response to renal mechanoreceptor (MR) stimulation. We have examined whether calcitonin gene-related peptide activates renal pelvic sensory receptors and whether such activation contributes to renal chemoreceptor stimulation. The calcitonin gene-related peptide receptor antagonist, h-CGRP (8–37) [0.01–10 μmol L^?1] dose-dependently decreased (29 ± 4–86 ± 13%, P < 0.01) the ipsilateral afferent renal nerve activity in response to the renal pelvic administration of calcitonin gene-related peptide (0.26 μmol L^?1). Renal pelvic perfusion with 900 m M NaCl also increased ipsilateral ARNA (23 ± 3% increase, P < 0.02) and contralateral urinary sodium excretion (13 ± 4% increase, P < 0.05). However, these responses to hypertonic NaCl were unaltered by h-CGRP (8–37). Renal pelvic perfusion with 1 or 10 μM h-CGRP (8–37) also failed to alter the ARNA responses to KCl (31.25, 62.5 and 125 m M ). These results indicate that there are sensory receptors in the renal pelvic area that are responsive to calcitonin gene-related peptide. The activation of these receptors elicits a contralateral natriuretic response. In contrast, the activation of renal calcitonin gene-related peptide receptors does not contribute to renal chemoreceptor activation.     

Endogenous CGRP protects retinal cells against stress induced apoptosis in rats

Acute stress can provoke apoptosis of the retina cells, and up-regulation of calcitonin gene related peptide (CGRP) in retina. However the role of CGRP in the pathology of the stress induced apoptosis of the retina cells is still elusive. The aim of this study was to investigate the endogenous of CGRP on retinal cell apoptosis induced by the stress of acute myocardial infarction induced by permanent coronary artery occlusion (CAO) in rats. The acute myocardial infarction model was established by ligating the left anterior descending branch of coronary artery in male Sprague-Dawley rats. The rats were randomized into two groups, the CGRP_8-37 group and a control group, pretreated with CGRP_8-37 (10⁻⁷ mol/L, 1 μL/g, intravenously injected), a specific antagonist of CGRP receptor, and 0.9% saline respectively, at 15 min prior to the CAO. The samples of the retina were collected at 3 h of CAO for the assays of TUNEL and caspase-3 activity respectively. The total apoptosis ratio of retinal cells in CGRP_8-37 group was significantly higher than that in the control group (P < 0.05). The capase-3 activity was significantly greater in the CGRP_8-37 group, compared with the control group (P < 0.05) at 3 h of CAO. The vacuolar degeneration of retinal ganglion cells was observed in the CAO animals. The results suggest that the endogenous CGRP may play a protective role in the retinal cell apoptosis induced by the stress of acute myocardial infarction.     

Tachykinins and calcitonin gene-related peptide as co-transmitters in local motor responses produced by sensory nerve activation in the guinea-pig isolated renal pelvis.

Electrical field stimulation of circular muscle strips from the guinea-pig isolated renal pelvis produces a frequency-dependent positive inotropic effect of the spontaneous contractions which is unaffected by atropine and guanethidine and abolished by tetrodotoxin or in vitro capsaicin desensitization. Omega conotoxin fraction GVIA markedly inhibited the response to low frequencies of stimulation but had only a partial or minor inhibitory effect at higher frequencies. Tachykinins produce a concentration-dependent positive inotropic effect, neurokinin A being more potent than substance P. On the other hand, rat alpha calcitonin gene-related peptide (CGRP) inhibited spontaneous contractions of the renal pelvis. MEN 10,376 a neurokinin A (4-10) analog, antagonized the positive inotropism produced by neurokinin A, without affecting the response to KCl, and suppressed the positive inotropic response produced by electrical field stimulation. In the presence of MEN 10,376, a negative inotropic response was produced by electrical field stimulation which was antagonized by the C-terminal fragment (8-37) of human alpha calcitonin gene-related peptide (hCGRP). hCGRP (8-37) antagonized the negative inotropic effect of exogenously administered CGRP without affecting inhibition by isoprenaline. Application of capsaicin (10 microM) produced a marked increase in the outflow of substance P-, neurokinin A- and CGRP-like immunoreactivities from the superfused guinea-pig renal pelvis. Substance P-, neurokinin A- and CGRP-like immunoreactivities were also detected in tissue extracts of the renal pelvis by radioimmunoassay. These experiments indicate that peptide release from peripheral endings of capsaicin-sensitive primary afferents represents the major type of nerve-mediated response affecting motility of the guinea-pig isolated renal pelvis. Tachykinins and CGRP act as physiological antagonists and the excitatory action of tachykinins prevails over the inhibitory action of CGRP. Local modulation of renal pelvis motility by sensory nerves could facilitate removal of irritants present in the urine, protecting the kidney during obstruction and ureteral antiperistalsis.     

Ontogeny of sensory nerves in the developing skeleton

Background: Previous studies have shown that the neuropeptide calcitonin gene-related peptide (CGRP) have an influence on osteoclastic bone resorption and that CGRP and substance P (SP), both wellknown markers for sensory neurons, behave as growth factors. Materials and Methods: The ontogeny of the sensory nerves in the hindlimb skeleton of the rat was studied from gestational day (GD) 15 to neonatal day (ND) 24 by immunohistochemistry. Neurofilaments and nerve terminals were labelled with protein gene-product 9.5 (PGP 9.5) and synaptophycin (SYN) respectively. Results: PGP 9.5 appeared at GD 15 and SYN at GD 19, both in the perichondrial tissue of the long bones. One week later, at ND 4 nerve fibre, immunoreactive to PGP 9.5 and SYN were observed within the bone organ. Sensory nerves, indicated by CGRP and SP, were first discerned at GD 18–19 in the periosteal tissue of the diaphyseal and metaphseal regions and in the bone organ at ND 4. Approximately at ND 6, vascular as well as non-vascular nerves extended into the metaphyses and at ND 8 into the epiphyses, concomitant with the first signs of mineralization. Conclusions: The study shows that a functional sensory nerve supply of the developing bone organ occurs immediatly prior to partus, apparently parallel with an increasing mineralization. The combined findings may indicate a sensory influence on developmental processes in the skeleton. © 1995 Wiley-Liss, Inc.     

Effects of hCGRP 8-37 and the NK1-receptor antagonist SR 140.333 on capsaicin-evoked vasodilation in the pig nasal mucosa in vivo

A novel pig in vivo model was used to study vascular effects of capsaicin, substance P and calcitonin gene-related peptide (CGRP) in the nasal mucosa and skin. An acoustic rhinometer was used to measure changes in nasal cavity volume, mainly representing changes in capacitance vessels in the vascular beds. The non-peptide NK₁-receptor antagonist SR 140.333 and the CGRP-receptor antagonist hCGRP 8-37 were used to investigate the role of substance P and CGRP, respectively, in capsaicin-evoked vasodilation mediated through activation of sensory C-fibre afferents. In this study we show that SR 140.333 is a potent inhibitor of substance P-induced vasodilation in the nasal mucosa whereas it has no effect on the capsaicin-evoked responses. Substance P only elicited a minor and shortlasting increase in superficial skin blood flow; this response, however, was completely blocked after administration of SR 140.333. Capsaicin-evoked vasodilation in the skin was slightly reduced by SR 140.333. CGRP-induced vasodilation in the nasal mucosa and skin was of much longer duration than the substance P-induced response, and was thus similar to the vascular effects mediated by capsaicin. hCGRP 8-37 significantly reduced both the CGRP- and capsaicin-mediated vasodilation in the nasal mucosa and the decrease of nasal cavity volume. Although the peak vasodilation in the skin in response to capsaicin, was unaltered by blockade of CGRP-receptors, the integrated response was significantly reduced by hCGRP 8-37. The present results show that vasodilatory responses to activation of afferent nerves in the pig nasal mucosa and superficial skin are mainly dependent on CGRP, while NK₁-receptor mechanisms seem to be of no or minor importance.     

Blood flow in the sciatic nerve is regulated by vasoconstrictive and vasodilative nerve fibers originating from the ventral and dorsal roots of the spinal nerves

Anesthetized rats were subjected to repetitive electrical stimulation of either the ventral or dorsal root of the spinal nerves between the 11th thoracic and 2nd sacral spinal segments. The response of nerve blood flow (NBF) in the sciatic nerve was examined using laser Doppler flowmetry. For all nerve fibers stimulation was for a 10–30-s period at a supramaximal intensity. (1) Stimulation of the T11-L1 ventral roots produced an increase in mean arterial pressure (MAP) and a biphasic NBF response was comprised of an initial increase and a subsequent decrease. The initial increase was a passive vasodilation due to the increase in MAP, while the following decrease in NBF resulted from active vasoconstriction of the vasa nervorum due to the activation of sympathetic nerves innervating the sciatic vasa nervorum. (2) Stimulation of the ventral root of the L6 segment produced an increase in NBF, even though MAP decreased. This increase in NBF was apparently mediated by activation of parasympathetic cholinergic vasodilators, because the response was abolished by i.v. injection of atropine, a muscarinic cholinergic receptor antagonist. (3) Stimulation of the dorsal roots between the L3 and S1 segments produced an increase in NBF, independent of changes in MAP. This increase in NBF appeared to be mediated by activation of a calcitonin gene-related peptide (CGRP) containing afferent fibers innervating the vasa nervorum, because the response was abolished by topical application of hCGRP (8–37), a CGRP receptor antagonist. In conclusion, NBF in the sciatic nerve is regulated by: (1) sympathetic vasoconstrictors exiting the ventral roots of the spinal cord via the T11-L1 segments; (2) parasympathetic vasodilators exiting the ventral roots of the spinal cord via the L6 segment; and (3) afferent CGRP containing vasodilators entering the dorsal roots of the spinal cord via the L3-S1 segments.     

Correlation of the directly observed responses of mesenteric vessels of the rat to nerve stimulation and noradrenaline with the distribution of adrenergic nerves

1. The effects of nerve stimulation and of the topical application of noradrenaline on arteries, capillaries and veins of the mesentery of the anaesthetized rat were examined by direct observation under a microscope. The distribution of adrenergic nerves to the vessels of the mesentery was studied using the fluorescence histochemical method.

2. Principal arteries, small arteries and terminal arterioles were all innervated by a network of adrenergic fibres and they all constricted in response to the stimulation of paravascular nerves and to exogenous noradrenaline. Few adrenergic fibres accompanied the smaller, precapillary arterioles; these vessels did not respond to nerve stimulation, although they were constricted by concentrations of noradrenaline as low as 10^-10 g/ml.

3. The capillaries did not respond to nerve stimulation or to applied noradrenaline. All veins were constricted by noradrenaline, but only those veins greater than about 30 μm in internal diameter responded to nerve stimulation.

4. At stimulus frequencies greater than 4 Hz the flow of blood through the microvasculature usually ceased, although there was never complete closure of these vessels. The maximum constriction observed in principal arteries was usually between 50 and 70% of the control internal diameter, and in small arteries and terminal arterioles was between 40 and 65% of the control internal diameter.

5. It is concluded that the principal arteries and small arteries of the mesenteric vasculature are important in the control of blood flow through this vascular bed during sympathetic stimulation and following topical application of noradrenaline, and that the precapillary arterioles are important vessels determining the rate of blood flow through the capillary bed under resting conditions.

     

Altered temperature and taste responses from cross-regenerated sensory nerves in the rat's tongue

1. The chorda tympani nerve, which innervates the front of the rat tongue, was found to be much less responsive to tongue cooling than the IXth nerve, which innervates the back of the tongue. The two nerves also differed in their relative responsiveness to various taste chemicals.

2. Through cross-union the IXth nerve was made to innervate the front of the tongue, and in other rats the chorda tympani nerve the back of the tongue.

3. After an average of 15 post-operative weeks, electrophysiological recordings of whole nerve action potential discharges were made from normal, control regenerated, and cross-regenerated nerves. Cooling, and chemical and mechanical stimulation of the tongue demonstrated that the control regenerated and cross-regenerated nerves had established functional connexions.

4. Neither the response to cooling nor the relative taste responses were altered by either of two types of control chorda tympani nerve regeneration.

5. In contrast, the cross-regenerated chorda increased its responsiveness to tongue cooling and the cross-regenerated IXthe nerve lost much of its responsiveness to cooling.

6. Cross-regeneration also caused the relative taste responses to change and appear quite similar to the responses obtained from the nerve which normally innervated that tongue region (e.g. the cross-regenerated IXth nerve responded like a chorda tympani nerve).

7. It is suggested that the sensory response evoked in the chorda tympani and IXth nerves by tongue cooling or taste stimulation is at least partially dependent upon the character of the tongue tissue in which the nerve terminates—the epithelium at the front differs from that at the back of the rat tongue.

8. These results rule out the following two hypotheses: (a) that the nerve ending itself functions as a taste receptor in direct contact with applied chemicals and yet is uninfluenced by the character of the tissue in which it terminates, (b) that assuming the taste bud cells are an integral part of the receptive process, the taste nerve ending determines the chemical specificity of the taste cell which it induces, without any previous modification of the nerve by the tissue in which it terminates.

     

The functional organization of the parasympathetic secretory innervation of the submandibular gland

1. In cats the hypoglossal and chorda-lingual nerves were cut and their central ends sewn to the peripheral end of the latter nerve. At monthly intervals the sensitivity of the submandibular gland to a secretory agent was then estimated.

2 When the supersensitivity resulting from the decentralization of the gland had been lowered to a constant level acute experiments were made. Electrical stimulation of both nerves caused secretion, which was abolished by ganglion blocking drugs and atropine.

3. Simultaneous stimulation of the nerves at frequencies subliminal for each nerve did not cause any secretion.

4. Stimulation of one nerve at a subliminal frequency did not increase a flow of saliva evoked by stimulation of the other nerve. However, as soon as the secretory threshold was reached, the flow was markedly accelerated, provided it was submaximal.

5. In many cases stimulation of the regenerated chorda fibres at a high frequency caused a flow corresponding to the maximal secretory capacity of the gland. In some experiments this was true for the hypoglossal fibres also.

6. The functional organization of the parasympathetic secretory innervation of the submandibular gland of the cat is discussed in the light of these observations.

     

Vascular responses of the spleen to nerve stimulation during normal and reduced blood flow

1. The splenic artery flow, the splenic weight and the arterial blood pressure were recorded in cats anaesthetized with sodium pentobarbitone.

2. Oscillations in splenic artery flow and splenic weight were observed. Following occlusion and release of the splenic artery, there was a brief increase in flow to above the pre-occlusion level and the oscillations in flow were greatly increased in amplitude. It is suggested that the brief increase is a consequence of the reduction of arterial pressure and that the oscillations are due to synchronization of rhythmic activity of smooth muscle within the spleen.

3. Stimulation of the splenic nerves resulted in decreases in splenic artery flow and splenic weight. The size of the responses varied with the frequency of stimulation and maximum responses in both flow and weight were obtained at about 3 impulses/sec.

4. After stimulation for 10 min, the splenic weight response was maintained while the flow response showed some recovery towards the control level.

5. When the splenic artery flow was reduced to about half the control level for periods up to 2 hr, the flow and weight responses to stimulation of the splenic nerves remained unchanged; the significance of this after a haemorrhage is discussed.

6. Intravenous administration of atropine or propranolol did not affect the responses to nerve stimulation. After phenoxybenzamine, nerve stimulation caused a smaller decrease in splenic weight, while the splenic artery flow increased to above the control level. This increase was unaffected by atropine but abolished by propranolol.