Sympathetic stimulation and blockade of the urinary bladder in cat

Ink J. Neuropharmocol., 1964.3, 241-251.

Pergamon Press.

Printed in Gt. Britain.

[l tables, 7 figs., 28 refs.1

SYMPATHETIC STIMULATION AND BLOCKADE OF THE URINARY BLADDER IN CAT E. B. SIGG and T. D. SIGG Geigy Research Laboratories,

Ardsley, N.Y.

Stunmary-Stimulation of hypogastric nerve contracts the urinary bladder. Exogenous norepinephrine and dopamine cause only an insignificant contraction or relaxation, whereas tyramine regularly contracts the bladder. Denervation or pharmacological sensitization by imipramine or cocaine does not alter the minor response to norepinephrine but the latter procedure enhances the effect of hypogastric stimulation. UML 491, a serotonin blocking agent, does not affect the hypogastric nerve-bladder response, whereas phentolamine has variable effects. Dihydroergotamine causes marked spontaneous contractions. Under certain conditions reserpine, syrosingopine and a-methylmetatyrosine diminish but never block the contraction induced by sympathetic stimulation. Choline-2,6-xylyl ether bromide markedly increases spontaneous contractions of the bladder, enhances the contractions to pelvic stimulation but irregularly diminishes those to hypogastric stimulation. Only guanethidine and bretylium block the hypogastric nerve-bladder response completely and selectively. Single pretreatment with syrosingopine (10 mg/kg i.v.), (Y-MMT (100 mg/kg i.v.) and guanethidine (15 mg/ kg i.v.) lowers the NE concentration of the bladder (18 hr) to approximately one third of that in controls, whereas the 5-HT content remains unchanged. The NE and 5-HT concentration in the untreated bladder is 1.13kO.48 and 0.20&0.03 pg/g respectively. Eserine augments and hexamethonium diminishes the sympathetically induced contraction.

INTRODUCTION

response of the urinary bladder to autonomic agents differs in many respects from other visceral organs. GYENMEK(1961) contended that the parasympathetic effector sites are functionally different from that of other organs whereas HENDERSONand ROEPKE (1935) favored the idea that a part of the parasympathetic motor innervation is noncholinergic. In regard to sympathetic-adrenergic influences on the bladder, the classical concept that hypogastric stimulation or catecholamines relax the detrusor muscle conflicts with numerous reports claiming contraction of the bladder wall. The early literature on these controversial findings has been summarized by INGERSOLLet al. (1954). Recent availability of agents which interfere with the release, synthesis or metabolism of the adrenergic transmitter made an analytical approach to investigate sympathetic nerve transmission to the bladder. The present study is concerned with the investigation of the effects of hypogastric nerve stimulation on the urinary bladder and the interference with sympathetic

THE

nerve

transmission

by various

agents.

METHODS Male cats weighing from 2 ~5to 3 -6 kg were used. All operative procedures were carried out under ether anesthesia. Tracheotomy and spinalization at Cl with mechanical destruction of the brain stem was performed. In some animals the spinal cord was pithed at 241

242

E. B. SIGG and T. D. SIGG

different levels. After midline incision the bladder was exposed, ureters tied and a polyethylene cannula inserted into the bladder through the urethra. Polyethylene cannulas were inserted into the carotid artery for blood pressure monitoring, the right iliac artery for retrograde injections into the abdominal aorta, and the femoral vein for intravenous administration of test drugs. The pelvic plexus on one side and both hypogastric nerves were dissected, ligated centrally, or in some instances, cut. Shielded bipolar platinum electrodes were inserted on their peripheral end. The bladder was then emptied and refilled with lo-15 ml of warm 0.976 saline solution. The exposed visceral organs were covered with moist gauze pads and body temperature was maintained by a heating lamp. Electrical stimulation was carried out alternatingly every 3-5 ‘min by means of two Grass SD-5 stimulators. The stimulus intensity was maximal (3-12 V), the frequency varying from 5 to 60 c/s. The individual pulse duration was 2 msec and the duration of the pulse train 5-30 sec. The carotid artery and bladder catheters were connected to suitable Statham pressure transducers, the output of which was fed into an Offner Dynograph Type R. For comparison in some experiments, the nictitating membrane was connected to a force displacement transducer (Grass FT 03) to measure the contractile response to electrical stimulation of the postganglionic cervical sympathetic nerves. Several experiments were performed in which a cannula was tied into the urethra distal to the internal sphincter. The ureter was cannulated to permit filling of the bladder, and pressure changes were recorded through a #24 needle inserted through the fundus of bladder. The following compounds were injected either retrograde into the iliac artery or into the femoral vein, as specified in the text. The number of experiments done with each drug is indicated in parentheses. The dose range used is stated in the text. Acetylcholine chloride (ACh) (9) atropine sulfate (2) bretylium tosylate (3), choline-2,6-xylyl ether bromide (TM 10) (4), cocaine HCl (l), dihydroergotamine (DHE 45) (2), dopamine (6) eserine (3) guanethidine (7) hexamethonium (C 6) (9), 5-hydroxytryptamine creatinine sulfate (5-HT) (29) imipramine (6) cr-methylmetatyrosine (cr-MMT) (5), norepinephrine (NE) (24), phentolamine (3), reserpine (8), syrosingopine (3) tyramine HCl (5) UML 491 (3). In some of the experiments involving reserpine, imipramine and guanethidine, the cats were acutely adrenalectomized. Experimentation was started not earlier than 1 hr after cessation of ether. Control responses were established for a period of 4-1 hr before injection of any test drugs. NE and 5-HT concentrations in the bladder tissue were spectrofluorometrically determined according to the method of BOGDANSKI et al. (1956) as subsequently modified by SHORE and OLIN (1958). The bladders were carefully freed of connective tissue and then cut in half to allow separate estimations of NE and 5-HT in the area of the fundus and of the trigone. The tissues were quick-frozen and kept for not more than 3 days before extraction. RESULTS In all experiments electrical stimulation of the hypogastric nerve elicited a contraction* of the bladder, the maximal response as a rule being less than that elicited by pelvic nerve stimulation. In contrast to the parasympathetically evoked response (P-response) the * Contraction as used in this text is defined as increase in intraluminal

pressure

at a given volume.

243

Sympathetic stimulation and blockade of the urinary bladder

contraction induced by sympathetic stimulation (H-response) declined rapidly before termination of stimulation and was sometimes followed by a relaxation of not more than one tenth of the amplitude of contraction. The character of these responses was not changed by pithing of the spinal cord. The maximal contractile response to hypogastric and pelvic stimulation was attained at 20 c/s. At 60 c/s the H-response was still close to maximal whereas the P-response was diminished (Fig. 1). Interindividual variations of threshold for eliciting the H-response were great while the P-response threshold varied less. During the second and third hour of experimentation both responses began to diminish spontaneously, the H-response usually to a greater extent than the P-response. When micturition was not possible because the cannulated urethra was connected to the pressure transducer, increasing the intravesicular volume diminished the response to pelvic stimulation whereas the H-response remained near control amplitudes. For this reason low bladder volumes (lo-15 ml) were used as standard. On the other hand, if pressure was measured through a hypodermic needle inserted in the bladder with the urethra unobstructed, increasing volumes (10-60 ml) caused little change in either the P- or H-response. Micturition could then sometimes be elicited by pelvic stimulation but not by bypogastric stimulation. The contraction resulting from maximal unilateral stimulation of the pelvic nerve was, however, never sufficient to achieve complete emptying of the bladder. It was of interest to note that, in confirmation of the observation of RUCH (1960), neither parasympathetic nor sympathetic stimulation caused either relaxation or active opening of the internal sphincter.

--_________

HYPOGASTRIC

-

___

5 cps 20

cps

60

cps

N.

FIG. 1. Effect of maximal pelvic and hypogastric stimulation at various frequencies for 30 set on intraluminal bladder pressure in spinalized cat.

H

E.B.SIGG and T. D. SIGG

244

In contrast to the hypogastric stimulation contraction, the presumed neurohumoral sympathetic transmitter, NE, and its precursor, dopamine, when injected in intra-arterial doses of 4-12 pg and l-10 mg respectively, caused either a small relaxation, or, infrequently, a slight contraction not exceeding 10 per cent of the maximal electrically induced response. Increasing volumes from 10 to 90 ml did not alter the response to these amines. On the other hand, tyramine (3-10 mg i.a.) induced a moderate contraction. S-HT in intra-arterial doses of 2-8 pg evoked a long lasting (lo-15 min) major contraction with rhythmic oscillations in pressure riding on the slowly declining phase (Fig. 2).

mmH20 100

50

0

A

4~~g NE i.0.

8mNE i.0.

3mg Do i.0.

FIG.2.Effect of norepinephrine, dopamine, tyramine and 5-hydroxyt~ptam~e on urinary bIadder of spinalizedcat. Pressure changes in response to intra-arterial injection of norepinephrine, dopamine, tyramine and 5-hydroxytryptamine. Calibrationsfor all tracings in mm X&O.

Adrenergic “sensitization” of the bladder by 500 &g/kg of imipra~ne or 1 mgikg of cocaine, as well as denervation by removal of both hypogastric nerves including their inferior mesenteric ganglia 2 weeks prior to the experiment, did not uncover a contractile response to exogenous NE as is observed in the comparably treated nictitating membrane. However, the two chemical “sensitizing” procedures led to an enhancement of the bladder response to hypogastric stimuIation (Fig. 3). In addition, cocaine and imipramine themselves increased the bladder tone slightly. UML-491, a 5-HT antagonist, did not affect the H-response in doses of lo-30 pgjkg, i.v., but progressively diminished and, at 100 pg,‘kg or more, blocked the 5-HT induced contraction of the bladder. Adrenolytic agents exerted variable effects. DHE (2-5 mg/kg i.v.) caused such strong, rhythmic contractions and rise in tone that responses to electrical stimulation of the hypogastric nerve could not be determined against this background of activity. PhentoIamine, (2-5 mgikg i.v.), while not affecting the bladder per se, generally decreased the H- and P-response. Occasionally, phentolamine increased the H-response.

245

Sympathetic stimulation and blockade of the urinary bladder

\. tt

L t

, I

Img

t

t

A s&gkg~

i

I

kg

4 ml”.

IMIPRAMINE

COCAINE I.“.

tP

H 1.~.

3. Effect of cocaine and iminramine on bladder tone and contraction evoked by pelvic (P) and cypogastric (H) nerve stimulation.. Note: Only responses to hypogastric nerve stimulation are shown in the case of the cocaine experiment.

FIG.

Further identification of the mechanism of hypogastric nerve-bladder transmission was attempted with the help of agents known to interfere with the synthesis or release of biogenic amines. Reserpine (5 mg/kg) increased the H-response by 4-24% during the first 90 min after injection, diminishing during the following 2 hr by not more than 20 per cent below control. On the other hand, the P-response diminished steadily and markedly after injection. After pretreatment with syrosingopine for 3 days at a dose of 3 x3 mg/kg i.p. (one experiment) and for 18 hr with 3-10 mg/kg i.v., the nictitating membrane and bladder response to stimulation of their respective postganglionic sympathetic nerve supply appeared diminished. The latter treatment with a single dose of 10 mg/kg i.v. diminished bladder NE content to approximately one third of that of the control whereas the 5-HT content remained the same (Table 1). During i.v. infusion of NE at the rate of 5 pg/min for 28 min, both responses were augmented (Fig. 4). This augmentation could not be demonstrated after pretreatment with guanethidine.

TABLE

Agent Control Guanethidine a-MMT Syrosingopine

1.

NOREPINEPHRINE AND 5-HYDROXYTRYPTAMINE BEFORE AND AFTER PRETREATMENT WITH VARIOUS

Number of samples

CONTENT OF URINARY BLADDER CATECHOLAMINE DEPLETORS

Number of experiments

Norepinephrine pg/g i standard deviation

5-Hydroxytryptamine I*g/g f standard deviation

Pretreatment dose

Hr before sacrifice

-

-

6

3

1.13 I-t 0.48

0.20 zt 0.03

15 mg/kg i.v.

18

6

3

0.36 f 0.15

0.24 & 0.02

100 mg/kg i.v.

18

6

3

0.43 + 0.16

0.24 rt 0.02

10 mg/kg i.v.

18

6

3

0.30 + 0.02

0.19 * 0.03

246

E. B. Srcc and T. D. SIGC

mmtlg

1.50

100

BL I

NM

NE 5pg

i.v. min.

FIG. 4. Effect of intravenous norepinephrine infusion on blood pressure, bladder response to hypogastric nerve stimulation @HJ and nictitating membrane contraction to postganglionic cervical sympathetic nerve stimulation (NM) in acute spinalized cat, pretreated with 3 mg/kg i.p. of reserpine 18 hr prior to experiment. Recordings in centre column were taken 15 min after the beginning of norepinephrine infusion; the tracings in the third column were recorded 15 min after termination of norepinephrine infusion. a-MTT (100 mg/kg i.v.) diminished the P- and H-response at the same rate and to about the same degree. An increase in spontaneous bladder activity and a slow rise in nictitating membrane tone and in blood pressure was noted. In contrast to the acute depressing effect of a-MMT on bladder transmission, the nictitating membrane contraction induced by postganglionic sympathetic stimulation was enhanced as was the vasopressor response to NE. Pretreatment with a single intravenous dose of a-MMT (100-300 mg/kg) 18 hr before experimentation did not indicate any impairment of hypogastric and pelvic transmission, although the NE content in the bladder was diminished considerably (Table 1). TM 10, given acutely in doses of lo-20 mg/kg i.v., increased the bladder tone immediately after injection and caused sustained rhythmic contractions of the bladder, An initial vasodepressor response was followed by a long lasting rise in blood pressure. The P-response was enhanced throughout the observation period (160 min after injection). The H-response diminished irregularly in three experiments and increased markedly in one. In contrast, the nictitating membrane response to postganglionic stimulation was completely blocked (Fig. 5). Bretylium (3-10 mg/kg i.v.) and guanethidine (I-10 mg/kg i.v.) were the only two compounds which completely and selectively blocked the H-response

Sympathetic stimulation and blockade of the urinary bladder

BLADDER

i

t

H

P

:I

!‘b

NICT. MEMBR.

4

NM

t

NM

t

t

IOmrj kg TM 10

NM

I”

FIG. 5. Effect of intravenous administration of TM 10 on responses of urinary bladder and nicitating membrane (contraction) to pelvic (I’), hypogastric (H) and postganglionic cervical sympathetic nerve stimulation. Immediate effect after injection and tracings taken 15 min after injection are shown.

PELVIC

STIMULATION

HYPOGASTRIC

STIMULATION

FIG. 6. Effect of intravenous injections of bretylium and guanethidine on contractile response of bladder to pelvic and hypogastric stimulation. C=control responses. The records are retraced on a compressed time scale.

247

E. B. SIGG and T. D. SIGG

248

and nictitating membrane contraction to sympathetic stimulation in acute experiments (Fig. 6). The blood pressure and nictitating membrane tone rose immediately after injection. Pretreatment with guanethidine for one (lo-15 mg/kg i.v.) and three (3 x 15 mg/kg i.v.) days also caused selective block of the bladder and nictitating membrane response to sympathetic stimulation, leaving P-response of the bladder and the vagal vasodepressor response intact. The NE concentration in the bladder was diminished to one third of that of controls while the 5-HT was not altered. The possible existence of a cholinergic component in hypogastric nerve-bladder transmission was then tested. Eserine (100-300 pg/kg i.v.) enhanced the H-response and the P-response. Subsequent injection of 2 mg/kg atropine antagonized the augmentation induced by eserine and reduced the H-response markedly (Fig. 7). While atropine (2 mg/kg) per se had no effect on the untreated H-response, C, (3-10 mg/kg i.v.) depressed it somewhat, but never as markedly as it depressed the P-response.

DISCUSSION

The present findings confirm the results of previous investigations (INGERSOLL et al., 1954): that stimulation of the hypogastric nerve contracts the urinary bladder and that the maximal contractile response is reached with stimulation frequencies of approximately 20 c/s as reported for other autonomic effector organs. The fact that the amplitude of contraction in response to hypogastric stimulation is less than that to pelvic stimulation may indicate that the sympathetic innervation is restricted. LANGWORTHY et al. (1940) claim that the detrusor muscle is devoid of sympathetic nerves and that sympathetic fibers reach only the representation of the ureteral muscles in the trigone. This assumption does not

mmHzO 100

50

0

tttttt\ P

H

c

1

H

lOOpg,

ES

FIG. 7. Effect of intravenous

kg

P

I

H

3OOpg/kg ES

P

I

t

H

L t P

2 mg;kg ATR

eserine administration (ES) followed by an intravenous injection of atropine (ATR) on the bladder response to hypogastric (H) and pelvic (P) nerve stimulation.

sympathetic

stimulation and blockade of the urinary bladder

249

hold in the light of our finding that the concentration of NE in the area of the fundus is not significantly different from that in the trigone area. That the contraction is not due to a constriction of blood vessels may be assumed for several reasons. First, KUNTZ and SACCOMANNO (1944) claim that the number of sympathetic fibers in the bladder is greater than that which would be expected necessary for inne~ation of blood vessels only. Secondly, injection of norepinephrine constricts the blood vessels without causing a bladder contraction. In contrast to the nictitating membrane, contraction of the bladder in response to electrical stimulation, particularly of the sympathetic nerve supply is not maintained during stimulation and is often followed by relaxation. The inability to maintain elevated pressure during stimulation is hardly due to exhaustion of transmitter substance since it even occurs during low frequency stimulation (5 c/s). The effect of exogenous NE or dopamine is equivocal and, even when ““sensitized” by surgical chronic denervation, cocaine or imipramine, the urinary bladder does not become responsive to these amines. However, under the latter two conditions the contraction response to hypogastric stimulation is slightly augmented. Tyramine, which at least partly exerts its effect through liberation of norepinephrine (TRENDELENBURC, 1961), contracts the bladder to some extent. Phentolamine produced variable results which are difficult to interpret at present. In some experiments it decreased the H- and P- nerve-bladder response and in others enhanced the H-response. BOYD et al. (1963) in experiments on various isolated nervesmooth muscle preparations, found that phentolamine blocks responses to adrenergic as well as cholinergic stimulation. On the other hand, OHL~Nand STROMBLAD (1963) observed an enhancement of the H-response in the isolated hypogastric nerve vas deferens preparation. Evidence that the sympathetically induced contraction is indeed due to release of adrenergic transmitter is accumulated by results obtained with agents interfering with the release or synthesis of catecholamines. During the early phase of reset-pine action, that is, during the release of catecholamines from tissue stores, spontaneous bladder activity and tone is increased and the H-response (but not the P-response) is enhanced. In the later stages, using syrosingopine pretreated animals, the H-response is still present although it seems diminished. (This diminution, however, could not be quantified as control responses in the same animal are unattainable). The improvement of the bladder and nictitating membrane response to sympathetic stimulation by NE infusion is in accordance with the finding of BURNand RAND(1958) that the NE releasing action of tyramine can be restored by replenishing the reserpine depleted stores with an infusion of NE. In acute preparations a-MMT reduces the H- and P-responses, while increasing blood pressure and tone of nictitating membrane. Pretreatment with a single dose of ol-MMT does not abolish the hypogastric nerve bladder transmission. Like reserpine, syrosingopine and ol-MMT produce a long lasting lowering of NE concentration in peripheral tissues believed to be due to an alteration in binding sites which are then unable to hold endogenousIy formed NE (HESS, 1962). The failure to obtain marked reduction or blockade of hypogastric nerve-bladder transmission with reset-pine, syrosingopine and a-MMT, may be the consequence of incomplete depletion of NE from the storage sites under study or a differential sensitivity of various endorgans to the action of these agents. VOGT (1963) calls attention to the resistance of hypogastric nerves of dog to the NE depleting effect of reserpine. Moreover, the NE content of untreated hypogastric nerves and bladder tissue is variable. According to OHL~Nand STROMBLAD (1963), denervation fails to deplete the vas deferens

250

E. B. SIGG and T. D. SIGG

of catecholamines. Differential release of NE by reserpine in various tissues has also been pointed out by TRENDELENBURG and WERNER(1962). As in the guinea pig vas deferens (BOYDet al., 1961) bretylium and guanethidine also block the bladder response to hypogastric stimulation. On the other hand, TM 10 causes marked rh~hmic contractions of the bladder and a potentiation of the parasympathetic stimulation effect. The latter two effects may be due to the muscarine-like properties of the drug (BAIN, 1960). Although these compounds have been claimed to release NE (SHEPPARDand ZIMMERMANN, 1959; COUPLANDand EXLEY,1957) the depletion produced does not account for adrenergic transmission failure (BAIN, 1960; KRONEBERG and SCHUMANN,1962), for, in the case of bretylium and guanethidine, the release of NE is slow, reaching the maximum depletion between four and eighteen hours after injection, whereas transmission blockade occurs within minutes. Moreover, while the NE concentration of the bladder is lowered to the same extent by pre-treatment with a-MMT, syrosingopine and guanethidine, sympathetic transmission is only blocked by the latter agent. ABERCROMBIE and DAVIES(1963) contend that guanethidine induces the transmission block by inhibiting the synthesis of NE, a conclusion also drawn by BAIN(1960) for TM 10. Infusion of NE in a guanethidine treated animal does not restore the vasoconstri~tor (MC~UBBIN et al., 1961) or bladder response to sympathetic stimulation, indicating that NE stores cannot be refilled as is the case with the reserpinized preparation. The question arises: does the sympathetic nerve-bladder transmission also involve a cholinergic mechanism as postulated by BURN and RAND (1960) for other postganglionic sympathetic neuroeffector systems. In apparent accordance with this hypo~esis are reports that hexamethonium diminishes or blocks hrpogastric nerve-vas deferens transmission (SJ~STRAND,1962) and eserine augments the contraction of the nictitating membrane to postganglionic cervical sympathetic stimulation (BURN et al., 1963) and of the guinea pig vas deferens (BOYD et al., 1960; BURN and WEETMAN,1963) and cat bladder to hypogastric nerve stimulation. However, BLAKELEYet al. (1963) pointed out that the end organ response does not necessarily reflect the amount of transmitter released. Thus, eserine and hexamethonium do not affect NE Iiberation by the splenic nerves. It is widely accepted that whenever hexamethonium blocks a response to nerve stimulation, there must be ganglionic synapses between the electrode and the responding smooth muscle cell. Abnormal location of ganglion cells in the course of presumably postganglionic sympathetic fibers (VANOVand VOGT (1963), preganglionic cholinergic fibers intermingled with postganglionic sympathetic nerves, and the cholinergic nature of certain postganglionic s~pa~eti~ fibers, may therefore be the reason for hexamethonium-induced diminution or blockade. In conclusion it may be stated that postganglionic sympathetic stimulation of the urinary bladder consistently elicits a motor response. Although pharmacological evidence using agents which augment or block the effect or synthesis of NE points to an adrenergic transmission mechanism, exogenous NE has no significant effect on the bladder, not even after drug induced ‘%ensitization”. This series of experiments supports the assumption that a cholinergic mechanism is involved in sympathetic nerve bladder transmission. R&sum&-La stimulation du nerf hypogastrique entraine la contraction de la vessie. La noradrenaline et la dopamine exogenes ne determinent qu’une contraction ou un relachement peu importants alors que la tyramine provoque systematiquement la contraction de la vessie. La denervation ou la sensibilisation pharmacologique par f’imipramine ou Ia cocaine n’alterent pas la response mineure a la noradr~naiine, mais ce demier pro&de renforce l’effet de la stimulation hypogastrique. Un blocant de la sbrotonine, I’UML 491, n’influence pas la reponse vesicale a la stimulation hypogastrique alors que la phentotamine engendre des effets variables.

Sympathetic stimulation and blockade of the urinary bladder

2.51

La dihydroergotamine induit des contractions spontanees intenses. Dans certaines conditions, la reserpine, la syrosingopine et l’cc-metatyrosine diminuent mais ne bloquent pas lacontraction provoquee par stimulation sympathique. La bromure ether de choline-2,6-xylyl augmente fortement les contraction spontanees de la vessie, renforce la contraction induitepar stimulation pelvienne, mais diminue dune man&e inconstante celles consecutives a stimulation hypogastrique. Seuls la guanethidine et la bretylium provoquent un blocage complet et selectif de la reponse vesicale a la stimulation hypogastrique. Une seule injection i.v. prealable de syrosingopine (10 mg/kg) d’a-MMT (100 mg/kg) et de guanethidine (15 mg/kg/ abaisse la conentration de NE de la vessie (18 h) a environ l/3 de la valeur des controles tandis que le taux de 5HT demeure inchange. La concentration de NE et de 5HT dans la vessie non traitee est respectivement de 1.13 & 0.48 et de 0.20 i_ 0.03 pg/g. L’eserine renforce et l’hexamethonium reduit la contraction d’origine sympathique. REFERENCES ABERCROMBIE, G. F. and DAVIES,B. N. (1963). The action of guanethidine with particular reference to the sympathetic nervous system. Brit. J. Pharmacol. 20: 171-177. BAIN, W. A. (1960). In Ciba Foundation Symposium on Adrenergic Mechanisms. Little, Brown, Boston, Mass. pp. 131-147. BLAKELEY,A. G. H., BROWN, G. L. and FERRY, C. B. (1963). Pharmacological experiments on the release of the sympathetic transmitter. J. Physiol. 167: 505-514. BOGDANSKI,D. F., PLETSCHER,A., BRODIE,B. B. and UDENFRIEND,S. (1956). Identification and assay of serotonin in brain. J. Pharmacol. 117: 82. BOYD,H., CHANG,V. and RAND, M. J. (1960). The anticholinesterase activity of some antiadrenaline agents. Brit. J. Pharmacol.

20: 74-82.

BOYD, H., CHANG, V. and RAND. M. J. (1961). The local anesthetic activity of bretylium in relation to its action in blocking sympathetic responses. Arch. int. Pharmacodyn. 131: 10-23. BOYD, H., BURNSTOCK,G., CAMPBELL,G., Jowsrr, A., O’SHEA, J. and WOOD, M. (1963). The cholinergic blocking action of adrenergic blocking agents in the pharmacological analysis of autonomic innervation. Brit. J. Pharmacol. 20: 418-435. BURN, J. H. and RAND, M. J. (1958). The action of sympathomimetic amines in animals treated with reserpine. J. Physiol. 144: 314-336. BURN, J. H. and RAND, M. J. (1960). Sympathetic postganglionic cholinergic fibres. Brit. J. Phnrmacol. 15: 56-66. BURN, J. H., RAND, M. J. and WEIN, R. (1963). The adrenergic mechanism in the nictitating membrane. Brit. J. Pharmacol.

20: 83-94.

BURN, J. H. and WEETMAN,D. F. (1963). The effect of eserine on the response of the vas deferens to hypogastric nerve stimulation. Brit. J. Pharmacol. 20: 74-82. COUPLAND,R. E. and EXLEY,K. G. (1957). Effects of choline 2:6-xylyl ether bromide upon the suprarenal medulla of the rat. Brit. J. Pharmacol. 12: 306-3 11. GYERMEK,L. (1961). Cholinergic stimulation and blockade on urinary bladder. Amer. J. Physiol. 201: 325-328.

HENDERSON,V. E. and ROEPKE,M. H. (1935). The urinary bladder mechanism. J. Pharmacol. 54: 408-414. HESS, S. M. (1962). The releasing action of a-methyl-3-hydroxyphenylalanine (a-methyl-meta-tyrosine). Arch. int. Pharmacodyn.

138: 584-590.

INGERSOLL,E. H., JONES,L. L. and HEGRE, S. E. (1954). Urinary bladder response to unilateral stimulation of hypogastric nerves. J. Ural. 72: 178-190. KRONEBERG,G. and SCHUMANN,H. J. (1962). Untersuchungen zum wirkungs-mechanismus des Guanethidins. Arch. exp. Path. u. Pharmak 243: 16-25. KUNTZ, A. and SACCOMANNO, G. (1944). Sympathetic innervation of the detrusor muscle. J. Urol. 51: 535-542. LANGWORTHY, 0. R., KOLB, L. C. and LEWIS,L. G. (1940). Physiology of micturition. Williams &Wilkins, Baltimore, Md. p. 232. MCCUBBIN, J. W., KANEKO,Y. and PAGE, I. H. (1961). The peripheral corticovascular actions of guanethidine in dogs. J. Pharmacol. exp. Therap. 131: 346-354. _ _ OHLIN, 0. and STROMBLAD, B. C. R. (1963). Observations on the isolated vas deferens. Brit. J. Pharmacol. 20: 299-306.

RUCH, T. C. (1960). In American Physiological Society. Handbook of Neurophysiology, Vol. 2. Williams & Wilkins, Baltimore, Md. p. 665. SHEPPARD, H. and ZIMMERMAN,J. (1959). Effect of guanethidine (SV-5864) on tissue catecholamines. Pharmacologist 1 (2): 69. SHORE,P. A. and OLIN, J. S. (1958). Identification and chemical assay of norepinephrine in brain and other tissue. J. Pharmacol. 122: 295-300.

E. B. SIGG and T. D. SIGG

252 SJOSTRAND,N. 0. (1962). Inhibition

of ganglionic blocking guinea pig vas deferens to hypogastric nerve stimulation. TRENDELENBERG,U. (1961). Modification of the effect of J. Pharnzacol. 134: 8-17. TRENDELENBERG,U. and WEINER, N. (1962). Sensitivity of cedures and agents. J. Phnrmacol. 136: 152-161. VANOV,S. and VOGT, M. (1963).

J. Physiol. 168: 939-944.

Catecholamine-containing

agents of the motor response of the isolated Acfa Physiol. Stand. 54: 306-315. tyramine by various agents and procedures. the nictitating

membrane

after various pro-

structures in the hypogastric nerves of the dog.