Effects of age and streptozotocin-induced diabetes on contents and effects of substance P and vasoactive intestinal polypeptide in the lower urinary tract of the rat.

The urinary bladder and urethral content of substance P and vasoactive intestinal polypeptide and the in vitro effects of the peptides on the bladder were studied at 6 weeks and 6 months of streptozotocin-induced diabetes in the rat. The results were compared with those obtained in age-matched control animals. Both short-term and long-term streptozotocin treatment induced a clearcut increase in bladder weight. Bladder substance P content was increased in both groups of diabetic animals but substance P concentration was similar in control and diabetic animals. Vasoactive intestinal polypeptide content was slightly higher in diabetic animals than in controls but vasoactive intestinal polypeptide concentration was significantly lower in the bladders from both short-term and long-term diabetic animals. The bladder contractile response to substance P was similar in all groups of animals and vasoactive intestinal polypeptide was found to be devoid of contractile or relaxatory effects in the rat bladder. No change in urethral weight was seen with diabetes. There were no clear-cut changes in the urethral contents or concentrations of substance P and vasoactive intestinal polypeptide. The study also enabled comparisons between younger (3 months) and older (9 months) rats. This comparison showed a decrease in the concentrations and contents of substance P and vasoactive intestinal polypeptide between young and older rats. The changes were seen in both the bladder and the urethra and were similar in diabetic and normal animals.     

Age-related changes in sensitivity of rat urinary bladder to autonomic agents

Experiments were done to determine if age-related changes occur in autonomic regulation of rat urinary bladder. The maximum contractile responses to acetylcholine were 63% and 15% greater in isolated bladders from 29-month and 17-month animals, respectively, as compared to 7-month animals. The amounts of [3H]quinuclidinyl benzilate bound to membrane preparations were 46% and 7% greater. In contrast, no age-related changes were observed in phenylephrine-induced contraction or in isoproterenol-induced relaxation of bladder. Thus, the urinary bladder of aged rats appears to develop increased sensitivity to cholinergic stimuli because of an increase in the number of muscarinic cholinergic receptors.     

Acetylcholine and substance P responsiveness of intestinal smooth muscles in streptozotocin diabetic rats

We found previously that in in vitro tube form preparations of isolated intestine of streptozotocin (STZ) diabetic rats, frequency of spontaneous intraluminal pressure waves was significantly reduced in duodenum when compared with normal controls. In order to elucidate further the diabetic intestinal disorders, we examined the frequency and amplitude of spontaneous length changes and contractile responses to acetylcholine (ACh) and substance P (sP) in isolated intestinal segments of normal and experimental diabetic rats. In comparison with normal controls, we could confirm the significantly decreased frequency of spontaneous length changes in isolated longitudinal and circular muscle preparations of diabetic duodenum (1 month after STZ injection). Furthermore, amplitude of spontaneous length changes was significantly decreased in circular muscle preparations of duodenum, jejunum, and ileum but not in colon nor in longitudinal muscle preparations. Dose-response curves revealed that both ACh and sP responses were significantly decreased in longitudinal and circular muscle preparations of diabetic duodenum, jejunum, and ileum but not in colon. Mechanisms of reduced contractility of diabetic intestinal smooth muscle in response to ACh and sP were discussed.     

Effects of age and streptozotocin-induced diabetes on contents and effects of substance P and vasoactive intestinal polypeptide in the lower urinary tract of the rat

Andersson , P. O., Fahrenkrug , J., Malmgren , A. & Uvelius , B. 1992. Effects of age and streptozotocin-induced diabetes on contents and effects of substance P and vasoactive intestinal polypeptide in the lower urinary tract of the rat. Acta Physiol Scand 144 , 361–368. Received 29 January 1 991 , accepted 11 October 1991. ISSN 0001–6772. Departments of Physiology, Clinical Pharmacology and Urology, University of Lund, Lund, Sweden and Department of Clinical Chemistry, Bispebjerg Hospital, Copenhagen, Denmark. The urinary bladder and urethral content of substance P and vasoactive intestinal polypeptide and the in vitro effects of the peptides on the bladder were studied at 6 weeks and 6 months of streptozotocin-induced diabetes in the rat. The results were compared with those obtained in age-matched control animals. Both short-term and long-term streptozotocin treatment induced a clearcut increase in bladder weight. Bladder substance P content was increased in both groups of diabetic animals but substance P concentration was similar in control and diabetic animals. Vasoactive intestinal polypeptide content was slightly higher in diabetic animals than in controls but vasoactive intestinal polypeptide concentration was significantly lower in the bladders from both short-term and long-term diabetic animals. The bladder contractile response to substance P was similar in all groups of animals and vasoactive intestinal polypeptide was found to be devoid of contractile or relaxatory effects in the rat bladder. No change in urethral weight was seen with diabetes. There were no clear-cut changes in the urethral contents or concentrations of substance P and vasoactive intestinal polypeptide. The study also enabled comparisons between younger (3 months) and older (9 months) rats. This comparison showed a decrease in the concentrations and contents of substance P and vasoactive intestinal polypeptide between young and older rats. The changes were seen in both the bladder and the urethra and were similar in diabetic and normal animals.     

Increased contractile responses to endothelin-1 and U46619 via a protein kinase C-mediated nifedipine-sensitive pathway in diabetic rat aorta

To determine how diabetes alters vasocontractile responses to endothelin-1 (ET-1) and the thromboxane A2-mimetic U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxyprostaglandin F2alpha) and to explore the possible mechanisms of the altered responses, contractions produced by these agonists were examined in aortic rings from rats with 8- to 12-weeks streptozotocin-induced diabetes in comparison with those from age-matched control rats. ET-1 (> or = 1 nM) and U46619 (> or = 100 nM) induced significantly greater contractions in diabetic aorta. The enhanced contractile responses of diabetic aorta to these agonists were abolished in the presence of 1 microM nifedipine, resulting in no significant difference in the maximum responses between control and diabetic aortas. Pretreatment with 1 microM calphostin C or 20 nM staurosporine caused marked reductions in contractions induced by ET-1 and U46619 in both control and diabetic aortas, and the difference in the maximum contractile responses to these agonists between control and diabetic aortas were eliminated by their treatment. These results suggest that chronic diabetes enhances aortic contractions induced by ET-1 and U46619 and the enhanced contractions are possibly due to an increased Ca2+ influx through transmembrane Ca2+ channels resulting from increased protein kinase C-activated process.     

Potentiation of serotonin-induced contractility of gastric fundus strips in lactating rats

Gastric fundus strips isolated from lactating, non-lactating and pregnant rats were used to obtain agonist-induced contractions in vitro and the mechanism of alteration in gastric contractility to serotonin during lactation was investigated. The gastric contractile responses to 5-HT expressed as area per unit mass of muscle (cm² g^-1 muscle) were 60–65 % greater in lactating rats compared with non-lactating rats. However, responses evoked by acetylcholine (ACh) or histamine were not different. The EC₅₀ values for 5-HT were not different in either group indicating that there was no alteration in the 5-HT affinity for its receptors in lactating rats. In pregnant rats, 5-HT-induced gastric responses were significantly (P < 0.05) lower than those of non-lactating rats. Pre-treatment of non-lactating rats with haloperidol (which increases plasma prolactin levels) enchanced the gastric contractile response to 5-HT (P < 0.05). On the other hand bromocriptine administration (which lowers plasma prolactin levels) in lactating or immature rats, decreased the contractile response to 5-HT significantly, while bath application of bromocriptine (0.1 μM) had no effect. Incubation of fundus strips in physiological solution containing prolactin (10 μg ml^-1) for 24 h decreased the 5-HT evoked contractions, but not the ACh evoked responses. The results in this study indicate that prolactin modulates the intrinsic contractile activity of the gastric smooth muscles to 5-HT.     

Bladder function in female rats: effects of aging and pregnancy

In vivo anesthetized cystometrograms and in vitro bladder tissue strip responses were examined in three groups of female rats: young virgins (3 month), older virgins (8 month), and retired breeders (8-9 month). Significant age-related in vivo changes were observed including greater resting pressures, but smaller voided volumes, void durations and void-to-void intervals in older versus young virgin rats. There were significant age-related changes in the in vitro responses. Greater peak and steady state contractions to high K+-modified Krebs (80 mM) depolarization were observed in young animals compared to older animals. Plus, young virgins exhibited greater sensitivity but smaller maximal, normalized contractions to acetylcholine (ACh) than older virgins. Diminished responses to adenosine-5'-triphosphate (ATP) were detected in young versus older virgin rats. Pregnancy-related changes were compared between retired breeders and their age-matched controls, older virgin rats. In vivo voided volumes were greater in the retired breeders than in the older virgins. Smaller in vitro steady state contractions to high K+-modified Krebs depolarization and smaller normalized contractions to maximal concentrations of ACh were observed in the retired breeders than in the older virgins. Retired breeders exhibited diminished relaxation responses to norepinephrine compared to older virgins. ATP produced greater dose-dependent responses and greater maximal contractions in the retired breeders compared to the older virgins. In conclusion, age-related changes were present even prior to the onset of senescence, and multiple pregnancies altered bladder function.     

The motor innervation of the rat urinary bladder

1. The post-ganglionic nerves visible in silver-impregnated sections of a normal rat bladder were absent 14 days after both pelvic ganglia had been ablated. After ablation of one ganglion the distribution of nerve trunks in either side of the organ was unchanged. Post-ganglionic axons from either side appear to distribute bilaterally.2. The acetylcholine (ACh) content of the rat bladder was reduced from control by 50% after the post-ganglionic nerves from one ganglion had degenerated. However, the ACh content in the two halves of the bladder sectioned along the mid line was the same after one nerve had degenerated.3. Motor responses of bladder preparations elicited during stimulation of both pelvic nerves were compared with responses elicited when each nerve was stimulated separately. In three-quarters of the animals the sum of the individual responses exceeded the response to combined nerve stimulation by no more than 20%. The functional overlap between the two groups of motor nerves to the bladder was therefore no greater than this amount in most animals.4. Motor responses of normally innervated bladder preparations elicited in vitro by transmural stimulation were compared with responses elicited after the post-ganglionic innervation from one side had degenerated. The mean response of bladders with half their innervation was 50-65% of the mean response of bladders normally innervated. The functional overlap by the two groups of nerves was found to be no greater than 15%.     

Cholinergic and serotonergic alterations in the rat hippocampus following trimethyltin exposure and fetal neural transplantation

The M2 receptor (M2-mAChR) is quantitatively the dominant muscarinic subtype in animal bladders. The alterations in its protein quantity and biosynthesis during diabetic cystopathy were investigated. Three-month-old male Wistar rats were divided into two groups: (1) 2-week-old diabetics; and (2) normoglycemic control rats. Diabetes was induced by single intravenous injection of 60 mg/kg streptozotocin. The amount of M2 receptor protein in the rat bladder body tissue was measured by Western immunoblotting using monoclonal antibodies. For determination of M2 muscarinic receptor mRNA in the bladder tissue, the method of Northern blotting was employed. The results of the Western immunoblotting showed that the amount of M2-mAChR protein in the diabetic bladder was significantly increased by 40.0 +/- 6.2% when compared with the control bladder (P < 0.05, n = 8). The Northern blotting demonstrated a 69.3 +/- 8.5% increase of the M2-mAChR mRNA in the diabetic bladder (P < 0.05, n = 8). The findings of the present study demonstrated an up-regulation of M2-mAChR biosynthesis in the diabetic urinary bladder. This phenomenon could lead to increased reactivity to acetylcholine and thus results in detrusor instability.     

Muscarinic supersensitivity in the rat urinary bladder after capsaicin pretreatment.

The effects of capsaicin on urinary bladder function have been investigated in adult rats. Ten days after capsaicin treatment immunocytochemical investigations showed a nearly complete disappearance of substance P (SP) and calcitonin gene-related peptide (CGRP) in all parts of the bladder. Recordings of micturition patterns and cytometrical investigations in conscious animals revealed no functional effects of capsaicin treatment. In-vitro experiments showed that the contractile response to substance P was similar before and after capsaicin treatment and CGRP exerted no contractile effects on the urinary bladder in either group of rats. The concentration-response curve to carbachol as well as the frequency-response curve to electrical stimulation were significantly shifted to the left in bladder muscle after capsaicin treatment. However, the maximal responses were similar in control and capsaicin-treated bladders. In the presence of scopolamine the maximal response to electrical stimulation was clearly lower in bladders subjected to capsaicin treatment than in controls. In conclusion, depletion of substance P and CGRP in the rat urinary bladder by capsaicin induced no supersensitivity to these peptides. However, the increased sensitivity to carbachol and to electrical stimulation seen after capsaicin treatment indicates the development of a supersensitivity to muscarinic receptor stimulation. Despite this supersensitivity in vitro no functional effects of capsaicin treatment were found in vivo.     

Urinary bladder instability induced by selective suppression of the murine small conductance calcium-activated potassium (SK3) channel

Small conductance, calcium-activated potassium (SK) channels have an important role in determining the excitability and contractility of urinary bladder smooth muscle. Here, the role of the SK isoform SK3 was examined by altering expression levels of the SK3 gene using a mouse model that conditionally overexpresses SK3 channels (SK3^T/T). Prominent SK3 immunostaining was found in both the smooth muscle (detrusor) and urothelium layers of the urinary bladder. SK currents were elevated 2.4-fold in isolated myocytes from SK3^T/T mice. Selective suppression of SK3 expression by dietary doxycycline (DOX) decreased SK current density in isolated myocytes, increased phasic contractions of isolated urinary bladder smooth muscle strips and exposed high affinity effects of the blocker apamin of the SK isoforms (SK1-3), suggesting an additional participation from SK2 channels. The role of SK3 channels in urinary bladder function was assessed using cystometry in conscious, freely moving mice. The urinary bladders of SK3^T/T had significantly greater bladder capacity, and urine output exceeded the infused saline volume. Suppression of SK3 channel expression did not alter filling pressure, threshold pressure or bladder capacity, but micturition pressure was elevated compared to control mice. However, SK3 suppression did eliminate excess urine production and caused a marked increase in non-voiding contractions. The ability to examine bladder function in mice in which SK3 channel expression is selectively altered reveals that these channels have a significant role in the control of non-voiding contractions in vivo . Activation of these channels may be a therapeutic approach for management of non-voiding contractions, a condition which characterizes many types of urinary bladder dysfunctions including urinary incontinence.     

Muscarinic supersensitivity in the rat urinary bladder after capsaicin pretreatment

The effects of capsaicin on urinary bladder function have been investigated in adult rats. Ten days after capsaicin treatment immunocytochemical investigations showed a nearly complete disappearance of substance P (SP) and calcitonin gene-related peptide (CGRP) in all parts of the bladder. Recordings of micturition patterns and cystometrical investigations in conscious animals revealed no functional effects of capsaicin treatment. In-vitro experiments showed that the contractile response to substance P was similar before and after capsaicin treatment and CGRP exerted no contractile effects on the urinary bladder in either group of rats. The concentration–response curve to carbachol as well as the frequency-response curve to electrical stimulation were significantly shifted to the left in bladder muscle after capsaicin treatment. However, the maximal responses were similar in control and capsaicin-treated bladders. In the presence of scopolamine the maximal response to electrical stimulation was clearly lower in bladders subjected to capsaicin treatment than in controls. In conclusion, depletion of substance P and CGRP in the rat urinary bladder by capsaicin induced no supersensitivity to these peptides. However, the increased sensitivity to carbachol and to electrical stimulation seen after capsaicin treatment indicates the development of a supersensitivity to muscarinic receptor stimulation. Despite this supersensitivity in vitro no functional effects of capsaicin treatment were found in vivo.     

Increased contractility to noradrenaline and normal endothelial function in mesenteric small arteries from the Goto-Kakizaki rat model of type 2 diabetes

Type 2 diabetes is associated with many circulatory manifestations, including alteration in endothelial function and hypertension. In this study we investigate the morphology and contractile response as well as the endothelial function of resistance arteries from the spontaneously diabetic Goto-Kakizaki (GK) rat, a model of lean type 2 diabetes expressing glucose intolerance. METHODS: Isolated mesenteric small arteries were investigated under isometric conditions in a wire myograph system using noradrenaline (NA) and the endothelium-dependent vasorelaxant acetylcholine (ACh). Media thickness was measured and media lumen ratio calculated. RESULTS: No apparent morphological difference was noted between the arteries from GK rats and control Wistar (CW) rats. When exposed to the maximal NA concentration used (30 microM), arteries from GK rats developed significantly more tension than arteries from CW rats. In the presence of indomethacin (a specific blocker of the COX synthase) and of L-NAME (an inhibitor of eNOS), the response to NA was still significantly greater in GK rat arteries. Under control conditions, arteries from both groups showed intact relaxation to ACh. After incubation with indomethacin and L-NAME, both groups showed a non-NO nonprostaglandin-dependent relaxation to ACh. This relaxation could be blocked by a combination of apamin and charybdotoxin. CONCLUSION: This study shows that mesenteric small arteries from the diabetic GK rat have increased contractile response to NA, along with a normal endothelial function and unaltered morphology.     

Role of different calcium pools in the contraction of respiratory smooth muscle

Contractions were evoked in isolated strips of rat trachea by potassium and acetylcholine (ACh) respectively. Extracellular calcium (Ca) was removed by EGTA and Ca influx was inhibited by diethylstilboestrol (DES). Contractions evoked by potassium were completely inhibited in the presence of EGTA or DES. ACh-induced contractions were rapidly abolished by EGTA whereas DES did not completely block these responses when a high concentration of ACh (10(-4) M) was used. Repeated activation by ACh in the presence of DES displayed two different phases of the contractile response; one initial brief contraction that decreased only slowly in the presence of DES, and a second phase with sustained contraction that was more readily depressed by DES. It is suggested that the smooth muscle in rat trachea is activated by Ca originating both intra- and extracellularly. As EGTA almost immediately blocked even the initial contractile response, the intracellular store is apparently located in or very close to the plasma membrane.     

Increase in choline acetyltransferase activity in surgically isolated postganglionic parasympathetic neurones of the urinary bladder of adult rats

In the urinary bladder of adult male rats the choline acetyltransferase activity in postganglionic neurones isolated from the central nervous system was shown to increase markedly and rapidly following section of the contralateral postganglionic neurones. The enzyme activity of the operated bladder was 32% of the control 3 days postoperatively, while at the last observation, 25 days postoperatively, it was 86%. As judged from additional studies on totally denervated bladders and on totally decentralized bladders the increase found in the enzyme activity was not due to ingrowth of nerves from outside or to unspecific acetylcholine synthesis neither was it due to increase in bladder wall tension or to increase in tissue mass.     

In vivo motor effects of loperamide on the rat urinary bladder.

Bladder motility recordings were performed in anaesthetized rats and the effect of the peripherally active opiate agonist loperamide on urinary bladder function was studied. Regional intra-arterial administration of loperamide (0.01-2 mg kg-1) induced weak bladder contraction per se. Loperamide caused an effective dose-dependent inhibition of bladder motility induced by regional injection of the receptor agonists acetylcholine (ACh) and substance P (SP), as well as by peripheral motor nerve stimulation (PNS). Pretreatment with naloxone (0.5 mg kg-1) partially antagonized the inhibitory action of loperamide on the nerve-mediated detrusor contraction. However, the depression of the motor responses induced by the receptor agonists ACh and SP was not influenced. It is suggested that the demonstrated inhibitory effect of loperamide on bladder motility is partially mediated by peripheral opioid receptors. The main non-opioid part of the inhibition might be a direct smooth muscle action.     

Vasomotor changes in isolated coronary arteries from diabetic rats

Contractile responses to prostaglandin F2 alpha, serotonin, noradrenaline, and potassium were examined in isolated intramyocardial arteries of Wistar rats 8 weeks after the induction of diabetes mellitus by administration of streptozotocin (STZ). The concentration-response curves obtained were compared with those noted in vessels both from age- and from weight-matched control rats. Light and electron microscopy did not reveal any major change in coronary artery wall thickness or morphology. There was no difference in the pattern of vasomotor responses between the two control groups. Contractile responses to prostaglandin F2 alpha, and potassium were significantly reduced, while contractile responses to serotonin and noradrenaline were unaltered in coronary arteries from diabetic rats. The vasomotor responses to noradrenaline and potassium showed a biphasic pattern in control vessels, i.e. contraction noted at high agonist concentrations was preceded by slight, but reproducible relaxation at lower concentrations. In diabetic vessels these relaxant responses were absent. The contraction produced by noradrenaline was markedly enhanced by the presence of propranolol in both diabetic and control vessels. Dilator responses to verapamil, diltiazem, nifedipine, papaverine and magnesium were studied in serotonin-precontracted coronary arteries; the concentration-response curves obtained by verapamil and diltiazem were shifted to the right in diabetic vessels. It appears justified to use vessels from age-matched rats as controls when vasomotor reactivity in coronary arteries from STZ-diabetic rats is investigated. The reduction in contractile responses to prostaglandin F2 alpha and potassium, and the reduction or lack of relaxant responses to noradrenaline, potassium, verapamil and diltiazem, in diabetic coronary arteries, indicate a selective modification of the coronary circulation by the diabetic disease.     

In vivo motor effects of loperamide on the rat urinary bladder

Bladder motility recordings were performed in anaesthetized rats and the effect of the peripherally active opiate agonist loperamide on urinary bladder function was studied. Regional intra-arterial administration of loperamide (0.01–2 mg kg^-1) induced weak bladder contraction per se. Loperamide caused an effective dose-dependent inhibition of bladder motility induced by regional injection of the receptor agonists acetylcholine (ACh) and substance P (SP), as well as by peripheral motor nerve stimulation (PNS). Pretreatment with naloxone (0.5 mg kg^-1) partially antagonized the inhibitory action of loperamide on the nerve-mediated detrusor contraction. However, the depression of the motor responses induced by the receptor agonists ACh and SP was not influenced. It is suggested that the demonstrated inhibitory effect of loperamide on bladder motility is partially mediated by peripheral opioid receptors. The main non-opioid part of the inhibition might be a direct smooth muscle action.     

Age-dependence of the spontaneous activity of the rat urinary bladder

Abnormal mechanical function of the bladder is manifested in a number of ways including higher frequency of involuntary detrusor contractions associated with reduced compliance of the bladder that is responsible for an increase in intraluminal pressure during filling. There are basically two ways to approach experimentally these problems: (1) by studying the neural control of the lower urinary tract function, and (2) by measuring the properties of smooth muscle cells in the bladder wall. Studies on smooth muscle function often do not take the origin of smooth muscle cells into account i.e., whether they were harvested from normal or overactive bladders. Although, this simplistic view may be beneficial to understanding the generation of the spontaneous activity of the bladder, however, it does not sufficiently explain the cell-to-cell propagation of the spontaneous smooth muscle activity. The spontaneous activity of smooth muscle is an important factor that works against the bladder compliance in the filling phase, and may inversely affect the neurally evoked response during micturition. The intensity of spontaneous activity is the age-dependent; it is high in neonatal bladders it is small or almost non-existent in adults and reemerges in older bladders. This review focuses on these age-dependent alterations of spontaneous bladder contractions and describes the possible mechanisms which may have important role in regulating the spontaneous contractions using the rat as an animal model.     

Altered urinary bladder function in mice lacking the vanilloid receptor TRPV1

In the urinary bladder, the capsaicin-gated ion channel TRPV1 is expressed both within afferent nerve terminals and within the epithelial cells that line the bladder lumen. To determine the significance of this expression pattern, we analyzed bladder function in mice lacking TRPV1. Compared with wild-type littermates, trpv1(-/-) mice had a higher frequency of low-amplitude, non-voiding bladder contractions. This alteration was accompanied by reductions in both spinal cord signaling and reflex voiding during bladder filling (under anesthesia). In vitro, stretch-evoked ATP release and membrane capacitance changes were diminished in bladders excised from trpv1(-/-) mice, as was hypoosmolality-evoked ATP release from cultured trpv1(-/-) urothelial cells. These findings indicate that TRPV1 participates in normal bladder function and is essential for normal mechanically evoked purinergic signaling by the urothelium.