- Email: [email protected]
Bladder Vasodilatation and Release of Vasoactive Intestinal Polypeptide from the Urinary Bladder of the Cat in Response to Pelvic Nerve Stimulation
0022-534 7/8? /l.S/33-0671$02.0G/O
Vol. 138, Sepcembe? Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright© 1987 by The Williams & WilkiEs Co.
BLADDER VASODILATATION AND RELEASE OF VASOACTIVE INTESTINAL POLYPEPTIDE FROM THE URINARY BLADDER OF THE CAT IN RESPONSE TO PELVIC NERVE STIMULATION P. 0. ANDERSSON,* S. R BLOOM, A. MATTIASSON AND B. UVELIUS From the Departments of Physiology and Urology, University of Lund, Sweden and the Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom
ABSTRACT
In the feline urinary bladder blood flow was determined by means of a direct blood flow measurement technique before and during pelvic nerve stimulation. Simultaneous sampling of venous blood from the bladder was performed, and the output of vasoactive intestinal polypeptide (VIP) was determined by means of radioimmunoassay. Maximal stimulation of the pelvic nerves led to a clearcut increase in intravesical pressure and a small but sustained increase of blood flow in the bladder wall. These changes were associated with a drastic increase in VIP output from the bladder, increasing from a control level of 0.2 fmol./min. to 15 fmol./min. during stimulation. The results suggest that VIP might be the neurotransmitter responsible for the vasodilatation in the feline urinary bladder in response to pelvic nerve stimulation. The discrepancy between the moderate blood flow increase and the pronounced increase in VIP-release might, however, indicate that VIP exerts its main effects elsewhere in the bladder than in the vascular bed, for instance the detrusor smooth muscle. (J. Ural., 138: 671-673, 1987) It is well known that the bladder contraction induced by pelvic nerve stimulation in most species is partly atropine resistant. 1 Nerve mediated events in the bladder vascular bed are, however, less well known. We have recently shown that pelvic nerve stimulation also induces a small but sustained increase in feline vesical blood flow. 2 The transmitter or neuromodulator responsible for this response has not been identified. There are, however, several putative mediators, among which are some neuropeptides. One of the neuropeptides present in the nerve terminals of the lower urinary tract and with known relaxatory influence on smooth muscle is VIP. 3 · 4 In a recent preliminary study5 in the cat VIP was shown to be released into the iliac veins after stimulation of parasympathetic efferents supplying the bladder. The present study was performed in cat using a direct blood flow measurement technique with simultaneous sampling of venous blood allowing analysis of feline vesical blood flow and output of VIP from the bladder in response to pelvic nerve stimulation.
distal ends mounted in ring electrodes. Square wave pulses (16 Hz, 10 v, 5 ms) found to cause maximum excitation, reflected by the intravesical pressure increase, were employed. It has previously been shown that parasympathetic nerve stimulation at 16 Hz induces maximal vasodilatation (and release of VIP) in the colon, the rectum, and the penis. 6 • 7 After the preparatory surgery the animals were left to equil ibrate for at least 30 min. before the effects of supramaximum pelvic nerve stimulation in periods of one to two minutes were investigated. Samples of arterial and venous effluent blood were collected for determinations of vasoactive intestinal polypeptide (VIP). The samples were collected in ice chilled tubes containing aprotinin (1000 KIU/mL blood;Bayer). To minimize the binding of VIP to the tubes, Minisorb test tubes (Nunc Inc., Denmark) were used. The samples were immediately centrifuged at +4C and the plasma stored at -20C until assayed. VIP concentrations were measured radioimmunoassay. 8 VIP output was calculated as the venous VIP concentration minus the arterial VIP concentration multiplied by the plasma flow (estimated from hematocrit determinations) and expressed as fmol./min. Statistical analysis is based on Student's t test and p values less than 0.05 were considered to be significant. Data are expressed as mean values ± SEM.
MATERIALS AND METHODS
The experiments were performed on male adult cats (mean weight 4.1 ± 0.2 kg., n = 6). Food, but not water was withheld for at least 14 hours before the experiment. The animals were anesthetised i.v. with chloralose (50 mg./kg.) and urethane (100 mg./kg.). A tracheal cannula was inserted to facilitate spontaneous respiration. The surgical procedure has been described in detail in a previous paper. 2 In brief, the abdomen was opened and a major vein draining the bladder was cannulated, whereas the other veins were ligated. The venous effluent blood flow was recorded via a photoelectric drop counter. The bladder was cannulated via the urethra for pressure recordings and the bladder volume was 10 to 20 ml. The pelvic nerves were dissected free 1 and the Accepted for publication May 20, 1987. * Requests for reprints: Dept. of Physiology, University of Lund, Solvegatan 19, S-223 62 Lund, Sweden. Supported by grants from the Swedish Medical Research Council (2210), the British Medical Research Council and the Medical Faculty, University of Lund, Sweden.
RESULTS
During the control period vesical blood flow averaged 0.24 ± 0.02 mL/min. (n = 6). Pelvic nerve stimulation induced an increase in bladder pressure and also a small but clearcut increase in bladder blood flow measured by direct recordings of venous outflow from the bladder. A typical pattern of response is illustrated in fig. L Urinary bladder pressure increased rapidly in response to pelvic nerve stimulation but the pressure quickly decreased during stimulation to a steady state level clearly below the peak response and bladder pressure returned to control levels again upon cessation of stimulation. Urinary bladder blood flow increased immediately after the onset of nerve stimulation (note the inverse ordinate scale) and the
671
672
ANDERSSON AND ASSOCIATES
blood flow increase was, in contrast to the bladder pressure response, maintained throughout the period of stimulation after which blood flow returned gradually to within the control level. Pelvic nerve stimulation at 16 Hz induced a significant increase in blood flow, from 0.24 ± 0.02 to a mean flow during the whole stimulation period of 0.32 ± 0.03 ml./min. (n = 6; p <0.01) (fig. 2). VIP output from the bladder was analyzed before and during pelvic nerve stimulation (fig. 2). During the control period there was only a small VIP output from the bladder (0.2 ± 1.4 fmol./min.; n = 6). During pelvic nerve stimulation the output increased markedly to 15 ± 9 fmol./min. (p <0.05). After stimulation VIP output returned to the control levels (not shown). Figure 3
ARTERIAL PRESSURE mmHg
200
150 100 0.1
~
0C\I I
E C)
~
100
<(
w
a:
(.)
z
w a:
::,
~ w a: a. a:
1-----i
1 min
50
w
0
D
<(
BLOOD FLOW ml•min-1 0.2
...J
co
peak response
0.3
0.5
URINARY BLADDER PRESSURE cm H 2 0
FIG. 3. Collected data from six experiments showing effects of pelvic nerve stimulation (16Hz) on urinary bladder pressure depicted as peak response and steady state response.
.........
STIMULATION
16 Hz
illustrates the peak pressure increase and the steady state increase in intravesical pressure obtained in these experiments. The increase in peak intravesical pressure averaged 95 ± 12 cm. H 2 0 and the increase in steady state intravesical pressure averaged 31 ± 4 cm. H20 (p <0.01).
200 100 0
j\_
DISCUSSION
FIG. 1. Original recordings of arterial blood pressure, bladder blood flow and bladder pressure from representative experiment in which pelvic nerves were stimulated at 16Hz for one min. Note inverse ordinate scale for blood flow recording.
control
0.50
pelvic nerve stimulation
20
~ 0
I::,
...J LL.
o 0 0
...J
Ill
0.. I-
0.25
steady state response
10
::,
0
a..
>
FIG. 2. Collected data from six experiments showing effects of pelvic nerve stimulation (16Hz) on bladder blood flow and output of VIP from bladder.
The present study on the feline urinary bladder shows that pelvic nerve stimulation results in an increased detrusor pressure, a small but sustained increase in bladder blood flow and a consistent increase in the output of VIP. It is well known that stimulation of parasympathetic nerves induces clearcut increases in VIP output in several other organs including the salivary glands,9 the colon, 10 the rectum 6 and the penis. 7 In this respect the feline urinary bladder behaved in a similar way to other parasympathetically innervated organs. In the above mentioned organs, parasympathetic nerve stimulation was found to induce a marked, non-cholinergic vasodilatation (five to 10-fold increase in blood flow). Therefore, VIP has been proposed to be a neurotransmitter released in response to parasympathetic nerve stimulation thereby causing vasodilatation in these tissues. On the other hand it has been found in the uterus, for example, that parasympathetic nerve stimulation leads to a marked increase in VIP output without any significant change in blood flow. 11 In the present study, pelvic nerve stimulation was found to increase vesical blood flow by only 30%. In view of these observations it is possible that VIP might be the neurotransmitter responsible for bladder vasodilatation in response to pelvic nerve stimulation. However, the discrepancy between the large increase in VIP output and the small increase in blood flow in response to pelvic nerve stimulation suggests that VIP might well have another primary target than the bladder vasculature. VIP is known to relax urinary bladder muscle strips and to reduce spontaneous contractions. 12·13 It
NERVE INDUCED BLADDER VASODKLATATWN AND VIP RELEASE
might therefore be speculated. that VIP release during pelvic nerve stimulation acts to decrease the tension of the bladder wall and hence the intravesical pressure during the later phase of excitation. Such a role for VIP, as a neurotransmitter or neuromodulator causing a relaxation of bladder smooth muscle, is corroborated by the preliminary observation that VIP concentration is reduced in unstable human bladder. 14
8.
REFERENCES
9.
7.
1. Langley, J. N. and Andersson, H.K.: The innervation of the pelvic
2. 3. 4.
5.
6.
and adjoining viscera. Part II: The bladder. J. Physiol., 19: 71, 1895. Andersson, P. 0., Bloom, S. R., Mattiasson, A. and Uvelius, B.: Changes in vascular resistance in the feline urinary bladder in response to bladder filling. J. Urol., 134: 1041, 1985. Gu, J., Polak, J.M., Blank, M.A., Terenghi, G., Morrisson, J. F. and Bloom, S. R.: The origin of VIP-containing nerves in the urinary bladder of rat. Peptides, 5: 219, 1984. Mattiasson, A., Ekblad, E., Sundler, F. and Uvelius B.: Origin and distribution of neuropetides Y-, vasoactive intestinal polypeptide- and substance P-containing nerve fibers in the urinary bladder of the rat. Cell Tissue Res., 239: 141, 1985. Blank, M. A., Anand, P., Lumb, B. M., Morrison, J. F. B. and Bloom, S. R.: Release of vasoactive intestinal polypeptide like immunoreactivity (VIP) from cat urinary bladder and sacral spinal cord during pelvic nerve stimulation. Dig. Dis. Sci., 29: 115, 1984. Andersson, P. 0., Bloom, S. R., Edwards, A. V., Jii.rhult, J. and
10.
11. 12.
13.
14.
673
Mellander, S.: Neural vasodilator control in the rectum of the cat and its possible mediation by vasoactive intestinal polypeptide. J. Physiol., 344: 49, 1983. Andersson, P. 0., Bloom, S. R. and Mellander, S.: Haemodynamics of pelvic nerve induced penile erection in the dog. Possible mediation by vasoactive intestinal polypeptide. J. Physiol., 350: 209, 1984. Mitchell, S. J. and Bloom, S. R.: Measurement of fasting and postprandial VIP in man. Gut., 19: 1043, 1978. Bloom, S. R. and Edwards, A. V.: Vasoactive intestinal polypeptide in relation to vasodilation in the submaxillary gland of the cat. J. Physiol., 300: 41, 1980. Fahrenkrug, J., Haglund, U., Jodal, M., Lundgren, 0., Olbe, L. and Schaffalitzky de Muckedell, 0. B.: Nervous release of vasoactive intestinal polypeptide in the gastrointestinal tract of cats: possible physiological implications. J. Physiol., 248: 291, 1978. Fahrenkrug, J. and Ottesen, B.: Nervous release of vasoactive intestinal polypeptide from the feline uterus: pharmacological characteristics. J. Physiol., 331: 451, 1982. Klarskov, P., Gerstenberg, T. and Hald, T.: Vasoactive intestinal polypeptide influence on lower urinary tract smooth muscle from human and pig. J. Urol., 131: 1000, 1984. Sjogren, C., Andersson, K.-E. and Mattiasson, A.: Effects of vasoactive intestinal polypeptide on isolated urethral and urinary bladder smooth muscle from rabbit and man. J. Urol., 133: 136, 1985. Gu, J., Restorick, J.M., Blank, M. A., Huang, W. M., Polak. J. M., Bloom, S. R. and Mundy, A. R.: Vasoactive intestinal polypeptide in the normal and unstable bladder. Br. J. Urol., 55: 645, 1983.
Vol. 138, Sepcembe? Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright© 1987 by The Williams & WilkiEs Co.
BLADDER VASODILATATION AND RELEASE OF VASOACTIVE INTESTINAL POLYPEPTIDE FROM THE URINARY BLADDER OF THE CAT IN RESPONSE TO PELVIC NERVE STIMULATION P. 0. ANDERSSON,* S. R BLOOM, A. MATTIASSON AND B. UVELIUS From the Departments of Physiology and Urology, University of Lund, Sweden and the Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom
ABSTRACT
In the feline urinary bladder blood flow was determined by means of a direct blood flow measurement technique before and during pelvic nerve stimulation. Simultaneous sampling of venous blood from the bladder was performed, and the output of vasoactive intestinal polypeptide (VIP) was determined by means of radioimmunoassay. Maximal stimulation of the pelvic nerves led to a clearcut increase in intravesical pressure and a small but sustained increase of blood flow in the bladder wall. These changes were associated with a drastic increase in VIP output from the bladder, increasing from a control level of 0.2 fmol./min. to 15 fmol./min. during stimulation. The results suggest that VIP might be the neurotransmitter responsible for the vasodilatation in the feline urinary bladder in response to pelvic nerve stimulation. The discrepancy between the moderate blood flow increase and the pronounced increase in VIP-release might, however, indicate that VIP exerts its main effects elsewhere in the bladder than in the vascular bed, for instance the detrusor smooth muscle. (J. Ural., 138: 671-673, 1987) It is well known that the bladder contraction induced by pelvic nerve stimulation in most species is partly atropine resistant. 1 Nerve mediated events in the bladder vascular bed are, however, less well known. We have recently shown that pelvic nerve stimulation also induces a small but sustained increase in feline vesical blood flow. 2 The transmitter or neuromodulator responsible for this response has not been identified. There are, however, several putative mediators, among which are some neuropeptides. One of the neuropeptides present in the nerve terminals of the lower urinary tract and with known relaxatory influence on smooth muscle is VIP. 3 · 4 In a recent preliminary study5 in the cat VIP was shown to be released into the iliac veins after stimulation of parasympathetic efferents supplying the bladder. The present study was performed in cat using a direct blood flow measurement technique with simultaneous sampling of venous blood allowing analysis of feline vesical blood flow and output of VIP from the bladder in response to pelvic nerve stimulation.
distal ends mounted in ring electrodes. Square wave pulses (16 Hz, 10 v, 5 ms) found to cause maximum excitation, reflected by the intravesical pressure increase, were employed. It has previously been shown that parasympathetic nerve stimulation at 16 Hz induces maximal vasodilatation (and release of VIP) in the colon, the rectum, and the penis. 6 • 7 After the preparatory surgery the animals were left to equil ibrate for at least 30 min. before the effects of supramaximum pelvic nerve stimulation in periods of one to two minutes were investigated. Samples of arterial and venous effluent blood were collected for determinations of vasoactive intestinal polypeptide (VIP). The samples were collected in ice chilled tubes containing aprotinin (1000 KIU/mL blood;Bayer). To minimize the binding of VIP to the tubes, Minisorb test tubes (Nunc Inc., Denmark) were used. The samples were immediately centrifuged at +4C and the plasma stored at -20C until assayed. VIP concentrations were measured radioimmunoassay. 8 VIP output was calculated as the venous VIP concentration minus the arterial VIP concentration multiplied by the plasma flow (estimated from hematocrit determinations) and expressed as fmol./min. Statistical analysis is based on Student's t test and p values less than 0.05 were considered to be significant. Data are expressed as mean values ± SEM.
MATERIALS AND METHODS
The experiments were performed on male adult cats (mean weight 4.1 ± 0.2 kg., n = 6). Food, but not water was withheld for at least 14 hours before the experiment. The animals were anesthetised i.v. with chloralose (50 mg./kg.) and urethane (100 mg./kg.). A tracheal cannula was inserted to facilitate spontaneous respiration. The surgical procedure has been described in detail in a previous paper. 2 In brief, the abdomen was opened and a major vein draining the bladder was cannulated, whereas the other veins were ligated. The venous effluent blood flow was recorded via a photoelectric drop counter. The bladder was cannulated via the urethra for pressure recordings and the bladder volume was 10 to 20 ml. The pelvic nerves were dissected free 1 and the Accepted for publication May 20, 1987. * Requests for reprints: Dept. of Physiology, University of Lund, Solvegatan 19, S-223 62 Lund, Sweden. Supported by grants from the Swedish Medical Research Council (2210), the British Medical Research Council and the Medical Faculty, University of Lund, Sweden.
RESULTS
During the control period vesical blood flow averaged 0.24 ± 0.02 mL/min. (n = 6). Pelvic nerve stimulation induced an increase in bladder pressure and also a small but clearcut increase in bladder blood flow measured by direct recordings of venous outflow from the bladder. A typical pattern of response is illustrated in fig. L Urinary bladder pressure increased rapidly in response to pelvic nerve stimulation but the pressure quickly decreased during stimulation to a steady state level clearly below the peak response and bladder pressure returned to control levels again upon cessation of stimulation. Urinary bladder blood flow increased immediately after the onset of nerve stimulation (note the inverse ordinate scale) and the
671
672
ANDERSSON AND ASSOCIATES
blood flow increase was, in contrast to the bladder pressure response, maintained throughout the period of stimulation after which blood flow returned gradually to within the control level. Pelvic nerve stimulation at 16 Hz induced a significant increase in blood flow, from 0.24 ± 0.02 to a mean flow during the whole stimulation period of 0.32 ± 0.03 ml./min. (n = 6; p <0.01) (fig. 2). VIP output from the bladder was analyzed before and during pelvic nerve stimulation (fig. 2). During the control period there was only a small VIP output from the bladder (0.2 ± 1.4 fmol./min.; n = 6). During pelvic nerve stimulation the output increased markedly to 15 ± 9 fmol./min. (p <0.05). After stimulation VIP output returned to the control levels (not shown). Figure 3
ARTERIAL PRESSURE mmHg
200
150 100 0.1
~
0C\I I
E C)
~
100
<(
w
a:
(.)
z
w a:
::,
~ w a: a. a:
1-----i
1 min
50
w
0
D
<(
BLOOD FLOW ml•min-1 0.2
...J
co
peak response
0.3
0.5
URINARY BLADDER PRESSURE cm H 2 0
FIG. 3. Collected data from six experiments showing effects of pelvic nerve stimulation (16Hz) on urinary bladder pressure depicted as peak response and steady state response.
.........
STIMULATION
16 Hz
illustrates the peak pressure increase and the steady state increase in intravesical pressure obtained in these experiments. The increase in peak intravesical pressure averaged 95 ± 12 cm. H 2 0 and the increase in steady state intravesical pressure averaged 31 ± 4 cm. H20 (p <0.01).
200 100 0
j\_
DISCUSSION
FIG. 1. Original recordings of arterial blood pressure, bladder blood flow and bladder pressure from representative experiment in which pelvic nerves were stimulated at 16Hz for one min. Note inverse ordinate scale for blood flow recording.
control
0.50
pelvic nerve stimulation
20
~ 0
I::,
...J LL.
o 0 0
...J
Ill
0.. I-
0.25
steady state response
10
::,
0
a..
>
FIG. 2. Collected data from six experiments showing effects of pelvic nerve stimulation (16Hz) on bladder blood flow and output of VIP from bladder.
The present study on the feline urinary bladder shows that pelvic nerve stimulation results in an increased detrusor pressure, a small but sustained increase in bladder blood flow and a consistent increase in the output of VIP. It is well known that stimulation of parasympathetic nerves induces clearcut increases in VIP output in several other organs including the salivary glands,9 the colon, 10 the rectum 6 and the penis. 7 In this respect the feline urinary bladder behaved in a similar way to other parasympathetically innervated organs. In the above mentioned organs, parasympathetic nerve stimulation was found to induce a marked, non-cholinergic vasodilatation (five to 10-fold increase in blood flow). Therefore, VIP has been proposed to be a neurotransmitter released in response to parasympathetic nerve stimulation thereby causing vasodilatation in these tissues. On the other hand it has been found in the uterus, for example, that parasympathetic nerve stimulation leads to a marked increase in VIP output without any significant change in blood flow. 11 In the present study, pelvic nerve stimulation was found to increase vesical blood flow by only 30%. In view of these observations it is possible that VIP might be the neurotransmitter responsible for bladder vasodilatation in response to pelvic nerve stimulation. However, the discrepancy between the large increase in VIP output and the small increase in blood flow in response to pelvic nerve stimulation suggests that VIP might well have another primary target than the bladder vasculature. VIP is known to relax urinary bladder muscle strips and to reduce spontaneous contractions. 12·13 It
NERVE INDUCED BLADDER VASODKLATATWN AND VIP RELEASE
might therefore be speculated. that VIP release during pelvic nerve stimulation acts to decrease the tension of the bladder wall and hence the intravesical pressure during the later phase of excitation. Such a role for VIP, as a neurotransmitter or neuromodulator causing a relaxation of bladder smooth muscle, is corroborated by the preliminary observation that VIP concentration is reduced in unstable human bladder. 14
8.
REFERENCES
9.
7.
1. Langley, J. N. and Andersson, H.K.: The innervation of the pelvic
2. 3. 4.
5.
6.
and adjoining viscera. Part II: The bladder. J. Physiol., 19: 71, 1895. Andersson, P. 0., Bloom, S. R., Mattiasson, A. and Uvelius, B.: Changes in vascular resistance in the feline urinary bladder in response to bladder filling. J. Urol., 134: 1041, 1985. Gu, J., Polak, J.M., Blank, M.A., Terenghi, G., Morrisson, J. F. and Bloom, S. R.: The origin of VIP-containing nerves in the urinary bladder of rat. Peptides, 5: 219, 1984. Mattiasson, A., Ekblad, E., Sundler, F. and Uvelius B.: Origin and distribution of neuropetides Y-, vasoactive intestinal polypeptide- and substance P-containing nerve fibers in the urinary bladder of the rat. Cell Tissue Res., 239: 141, 1985. Blank, M. A., Anand, P., Lumb, B. M., Morrison, J. F. B. and Bloom, S. R.: Release of vasoactive intestinal polypeptide like immunoreactivity (VIP) from cat urinary bladder and sacral spinal cord during pelvic nerve stimulation. Dig. Dis. Sci., 29: 115, 1984. Andersson, P. 0., Bloom, S. R., Edwards, A. V., Jii.rhult, J. and
10.
11. 12.
13.
14.
673
Mellander, S.: Neural vasodilator control in the rectum of the cat and its possible mediation by vasoactive intestinal polypeptide. J. Physiol., 344: 49, 1983. Andersson, P. 0., Bloom, S. R. and Mellander, S.: Haemodynamics of pelvic nerve induced penile erection in the dog. Possible mediation by vasoactive intestinal polypeptide. J. Physiol., 350: 209, 1984. Mitchell, S. J. and Bloom, S. R.: Measurement of fasting and postprandial VIP in man. Gut., 19: 1043, 1978. Bloom, S. R. and Edwards, A. V.: Vasoactive intestinal polypeptide in relation to vasodilation in the submaxillary gland of the cat. J. Physiol., 300: 41, 1980. Fahrenkrug, J., Haglund, U., Jodal, M., Lundgren, 0., Olbe, L. and Schaffalitzky de Muckedell, 0. B.: Nervous release of vasoactive intestinal polypeptide in the gastrointestinal tract of cats: possible physiological implications. J. Physiol., 248: 291, 1978. Fahrenkrug, J. and Ottesen, B.: Nervous release of vasoactive intestinal polypeptide from the feline uterus: pharmacological characteristics. J. Physiol., 331: 451, 1982. Klarskov, P., Gerstenberg, T. and Hald, T.: Vasoactive intestinal polypeptide influence on lower urinary tract smooth muscle from human and pig. J. Urol., 131: 1000, 1984. Sjogren, C., Andersson, K.-E. and Mattiasson, A.: Effects of vasoactive intestinal polypeptide on isolated urethral and urinary bladder smooth muscle from rabbit and man. J. Urol., 133: 136, 1985. Gu, J., Restorick, J.M., Blank, M. A., Huang, W. M., Polak. J. M., Bloom, S. R. and Mundy, A. R.: Vasoactive intestinal polypeptide in the normal and unstable bladder. Br. J. Urol., 55: 645, 1983.