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M R Ruggieri

Publications and source records attributed to M R Ruggieri.

At least 19 recordsLinked to original sources
PubMed2006

Regulation of bladder muscarinic receptor subtypes by experimental pathologies.

1 The M3 muscarinic receptor subtype is widely accepted as the receptor on smooth muscle cells that mediates cholinergic contraction of the normal urinary bladder and other smooth muscle tissues, however, we have found that the M2 receptor participates in contraction under certain abnormal conditions. The aim of this study was to determine the effects of various experimental pathologies on the muscarinic receptor subtype mediating urinary bladder contraction. 2 Experimental pathologies resulting in bladder hypertrophy (denervation and outlet obstruction) result in an up-regulation of bladder M2 receptors and a change in the receptor subtype mediating contraction from M3 towards M2. Preventing the denervation-induced bladder hypertrophy by urinary diversion prevents this shift in contractile phenotype indicating that hypertrophy is responsible as opposed to denervation per se. 3 The hypertrophy-induced increase in M2 receptor density and contractile response is accompanied by an increase in the tissue concentrations of mRNA coding for the M2 receptor subtype, however, M3 receptor protein density does not correlate with changes in M3 receptor tissue mRNA concentrations across different experimental pathologies. 4 This shift in contractile phenotype from M3 towards M2 subtype is also observed in aged male Sprague-Dawley rats but not females or either sex of the Fisher344 strain of rats. 5 Four repeated, sequential agonist concentration response curves also cause this shift in contractile phenotype in normal rat bladder strips in vitro, as evidenced by a decrease in the affinity of the M3 selective antagonist p-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD). 6 A similar decrease in the contractile affinity of M3 selective antagonists (darifenacin and p-F-HHSiD) is also observed in bladder specimens from patients with neurogenic bladder as well as certain organ transplant donors. 7 It is concluded that although the M3 receptor subtype predominantly mediates contraction under normal circumstances, the M2 receptor subtype can take over a contractile role when the M3 subtype becomes inactivated by, for example, repeated agonist exposures or bladder hypertrophy. This finding has substantial implications for the clinical treatment of abnormal bladder contractions.

Age Factors↗
PubMed2005

Bile duct ligation induced acute inflammation up-regulates cyclooxygenase-2 content and PGE2 release in guinea pig gallbladder smooth muscle cell cultures.

INTRODUCTION: This study examines hypotheses that BDL induces increased guinea pig gallbladder smooth muscle PGE2 release by up-regulation of COX-2. METHODS: BDL, Sham and Control Hartley guinea pig gallbladders were placed in cell culture, grown to confluence and underwent Western Blot analysis for smooth muscle cell content of COX-1, COX-2, Prostacylin Synthase, actin, caldesmon, vinculin, meta-vinculin and tropomyosin and were assayed for basal release of 6-keto-PGF(1alpha), PGE2 and TxB2 by EIA. RESULTS: BDL did not alter content of smooth muscle cytoskeletal proteins. BDL for 48 h increased smooth muscle cell release of PGE2 and 6-keto-PGF(1alpha) by 3-fold or more when compared to the Control and Sham groups. Western Blot analysis showed increased content of COX-2 in the BDL group. CONCLUSIONS: BDL for 48 h markedly increased endogenous guinea pig smooth muscle cell PG release, which was due to increased COX-2 synthesis.

Animals↗
PubMed2000

Role of neurokinin receptors in the behavioral effect of intravesical antigen infusion in guinea pig bladder.

PURPOSE: To characterize a guinea pig behavior model of bladder pain due to intravesical antigen infusion and to determine the role of neurokinin receptor subtypes in mediating this behavior. MATERIALS AND METHODS: The influence of subtype-selective neurokinin receptor antagonists on increased abdominal licking behavior in response to intravesical antigen infusion in guinea pigs immunized with ovalbumin (OA) was determined. RESULTS: Intravesical OA infusion for 30 minutes induced a significantly greater frequency (about 3-fold) of abdominal licking behavior than during either the 30 minutes pre-challenge or post challenge saline infusions. Treatment with IP capsaicin 7 to 10 days before OA challenge abolished the intravesical antigen-induced behavior. IP injection of the NK1 receptor antagonist CP 99994 (10 mg./kg. or 30 mg./kg.), 30 minutes pretreatment, inhibited the increase in the average number of abdominal licks during antigen infusion. The 30 mg./kg., but not the 10 mg./kg. dose increased the percent of animals showing antinociceptive activity (defined as 4 or less abdominal licks during the antigen infusion). The NK2 receptor antagonist SR 48968 reduced the antigen-induced abdominal licking behavior at IP doses of 3 and 10 mg./kg. but was ineffective at 1 mg./kg. The NK3 receptor antagonist SB 235375 (30 mg./kg., IP) did not reduce this behavior. CONCLUSIONS: These results suggest a role for activation of NK1 and NK2, but not NK3 receptors, by tachykinins released from capsaicin-sensitive nerves, in the increased abdominal licking behavior response of guinea pigs to intravesical antigen infusion.

Administration, Intravesical↗
PubMed1999

The effect of pregnancy and contractile activity on bladder muscarinic receptor subtypes.

In the rabbit bladder, pregnancy and prolonged bladder contractions decrease both muscarinic receptor density and contractile response, whereas newborns show enhanced muscarinic contractile response. Although the M(2) receptor predominates in rabbit bladder, we and others have shown that the affinity of a series of subtype selective muscarinic antagonists for inhibition of muscarinic agonist-induced contractions is most consistent with the pharmacologically defined M(3) receptor directly mediating smooth muscle contraction. Bladders from fetal rabbits, gravid rabbits, and male rabbits exposed to 4 hr of induced spontaneous contractions were used to determine whether changes in receptor density and contractility are due to a selective decrease in either the M(2) or M(3) muscarinic receptor subtype. To determine organ specificity, the heart and uterus were also studied. Gravid rabbits of 3 weeks' gestation and their fetal rabbits were studied. In male rabbits, bladder contractions were induced for 4 hr by ligating the catheterized penis at its base. Muscarinic receptor density and subtype distribution were determined by radioligand binding and immunoprecipitation. Receptor density was 24% lower in gravid bladder body, unchanged in gravid bladder base, 54% lower in gravid uterus, 115% higher in fetal bladders, and 34% lower after induced bladder contractions. Immunoprecipitation showed greater M(2) receptors than M(3) in all tissues studied, whereas M(l) and M(4) receptors were undetectable. There was no difference from control in the ratio of M(2) to M(3) receptor in any tissues except that a greater proportion of M(3) receptors was found in male vs. female bladders. Changes in contractile response to cholinergic stimulation in the gravid, fetal, and experimental detrusor instability model are associated with changes in total receptor density and not solely with changes in the M(3) receptor subtype that mediates bladder smooth muscle contraction. Neurourol. Urodynam. 18:511-520, 1999.

Animals↗
PubMed1999

Sex differences and role of nitric oxide in blood flow of canine urinary bladder.

Continuous measurements were made of bladder blood flow by laser Doppler flowmetry in anesthetized dogs during bladder filling and emptying. In both mucosa and muscle, perfusion was inversely proportional to intravesical pressure. There was significantly greater perfusion in the bladder mucosa of males than females at baseline and up to 10 cm water filling pressure but not in the muscle. Intra-arterial infusion of the nitric oxide synthase inhibitor NG-nitro-L-arginine produced a significant decrease in resting bladder perfusion in the mucosa only, with no differences seen in the response to intravesical pressure. Intra-arterial infusion of L-arginine produced a significant increase in the level of perfusion in the mucosa seen immediately after the bladder was drained. No changes were observed in muscle perfusion after L-arginine. These results suggest that the perfusion of the bladder mucosa differs by gender and is regulated differently than the bladder muscle, possibly related to the different function of the two layers.

Animals↗
PubMed1999

Selective alkylation of rat urinary bladder muscarinic receptors with 4-DAMP mustard reveals a contractile function for the M2 muscarinic receptor.

Our previous data indicate that M3 muscarinic receptors mediate carbachol induced bladder contractions. The data presented here were obtained by selective alkylation of M3 receptors with 4-DAMP mustard and suggest that the M2 receptor subtype may be involved in inhibition of beta-adrenergic receptor induced relaxation, therefore, allowing recontraction. Alkylation resulted in 85% of M3 receptors and 65% of M2 receptors unable to bind radioligand as demonstrated by subtype selective immunoprecipitation. Rat bladder strips subjected to our alkylation procedure contracted submaximally, and direct carbachol contractions were inhibited by antagonists with affinities consistent with M3 receptor mediated contraction. In contrast, the affinities of antagonists for inhibition of carbachol induced recontractions following isoproterenol stimulated relaxation in the presence of 90 mM KCl, indicated a contractile function for the M2 receptor that was not observed in control strips. In conclusion, these studies demonstrate a possible role for the M2 subtype in bladder smooth muscle contraction.

Alkylation↗
PubMed1999

Comparison of bladder blood flow in patients with and without interstitial cystitis.

PURPOSE: We compared bladder blood flow during filling and emptying in patients with and without interstitial cystitis, and correlated blood flow with symptoms in those with interstitial cystitis. MATERIALS AND METHODS: Bladder perfusion was measured using a dual channel endoscopic laser Doppler flow probe. Measurements were obtained in superficial and deeper vascular beds from the bladder mucosa at the trigone and back wall at baseline, at the volume of awake capacity, during 80 cm. water hydrodistention and after bladder drainage. American Urological Association symptom score was obtained preoperatively in interstitial cystitis patients. RESULTS: In all areas bladder perfusion decreased with filling in interstitial cystitis patients and increased in those without interstitial cystitis. There were no significant differences in response to emptying the bladder, as perfusion tended to increase in both groups. There was no correlation between bladder perfusion at baseline, or in response to filling or emptying with overall symptom score. CONCLUSIONS: Bladder perfusion decreases with bladder filling in patients with but increases in those without interstitial cystitis. The inability of the interstitial cystitis bladder to increase bladder blood flow with filling may be a reflection of other pathological processes in the bladder mucosa. The lack of correlation between blood flow and symptoms suggests that bladder ischemia alone cannot account for the symptoms in interstitial cystitis.

Adult↗
PubMed1998

Lack of effect following repeated in vivo exposure of the rabbit urinary bladder to urine from interstitial cystitis patients at low infusion volumes.

We reported previously that substances in interstitial cystitis urine, when infused into the rabbit bladder, induce changes that resemble bladders of interstitial cystitis (IC) patients. Here we report our investigation of the effect of additional molecular weight subfractions of IC urine and lower infusion volume in this rabbit bladder bioassay. Urine was pooled from symptomatic IC patients, asymptomatic IC patients (in remission), and normal volunteers. Two fractions of 20x concentrated urine were obtained for each of the 3 groups: a 10-100-kD fraction and a fraction > 100 kD but <0.22 microm. Six rabbits per group were infused twice per week with 6 ml of 1 of these 6 urine fractions or saline as a control. After 6 weeks, each rabbit was cystoscoped before and after hydrodistension, bladder capacity and urea permeability were determined, and the bladder was removed for histologic examination. A questionnaire revealed a significant difference (P < 0.01) regarding voiding symptom severity between symptomatic IC patients and both normal volunteers and IC patients in remission. There was no statistically significant difference among groups of rabbits in cystoscopic bladder appearance, bladder capacity, urea permeability, or bladder histology. If a urine-borne factor is in part responsible for IC symptoms, the rabbit bladder must be filled with urine to near capacity to be able to detect a difference between IC and normal urine in this rabbit bladder bioassay.

Administration, Intravesical↗
PubMed1998

Prejunctional M1 facilitory and M2 inhibitory muscarinic receptors mediate rat bladder contractility.

Subtype-selective muscarinic antagonists effects on carbachol-induced and electric field-stimulated contractility of rat bladder were compared in vitro. Schild plot analysis of cumulative carbachol dose-response curves in the presence of antagonists was consistent with M3-mediated bladder contractions. However, nerve-evoked contractions were inhibited 15% at 30 Hz (P < 0.01) by 10 nM pirenzepine (M1-selective antagonist), whereas 10 nM methoctramine (M2-selective antagonist) increased these contractions by 17% at 30 Hz (P < 0.01). Identical doses had no effect on carbachol-induced contractions, indicating prejunctional M1 facilitory and M2 inhibitory receptors. m1 Receptors could not be identified by subtype-selective antibodies, nor could the m1 transcript be identified by Northern hybridization. However, m1, m2, m3, and m4 transcripts were identified in rat bladder using the reverse transcriptase-polymerase chain reaction, providing support for the existence of the m1 subtype. In conclusion, strong evidence is provided for the existence of prejunctional M1 facilitory and M2 inhibitory and postjunctional M3 receptors modulating contractility in the rat urinary bladder.

Animals↗
PubMed1998

M2 muscarinic receptor contributes to contraction of the denervated rat urinary bladder.

In vitro bladder contractions in response to cumulative carbachol doses were measured in the presence of selective muscarinic antagonists from rats that had their major pelvic ganglion bilaterally removed. Denervation induced both hypertrophy and a supersensitivity of the bladders to agonist. The affinities in control bladders for antagonism of carbachol-induced contractions were consistent with M3-mediated contractions. Affinities in denervated bladders for 4-diphenlacetoxy-N-methylpiperidine methiodide (8.5) and p-fluoro hexahydrosilodifenidol (6.6) were consistent with M2-mediated contractions, although the methoctramine affinity (6.5) was consistent with M3-mediated contractions. Subtype-selective immunoprecipitation of muscarinic receptors revealed a 50% increase in total and a 60% increase in M2 receptor density with no change in M3 receptor density in denervated bladders compared with normal or sham-operated controls. This increase in M2 receptor density is consistent with the change in affinity of the antagonists for inhibition of carbachol-induced contractions and may indicate that M2 receptors or a combination of M2 and M3 receptors directly mediates smooth muscle contraction in the denervated bladder.

Animals↗
PubMed1997

Role of m1 receptor-G protein coupling in cell proliferation in the prostate.

The prostate gland from several animal species contains variable levels of muscarinic subtypes, but only the human prostate expresses significant levels of the m1 subtype. We studied muscarinic receptor activity in human benign prostatic hypertrophy (BPH) as well as several cell lines derived from prostate cancer. The BPH we studied expresses approximately 75% of the m1 receptor and undetectable levels of the other receptor subtypes whereas PC3 cells express only the m3 receptor subtype. DU145 and LnCaP cells express approximately equal levels of m1 and m3 receptor subtypes. Only the PC3 cells responded to carbachol with an increase in turnover of polyphosphoinositides, and none of the cell lines responded with effects on cAMP metabolism. Co-precipitation of receptors with heterotrimeric guanine nucleotide-binding regulatory proteins demonstrated interactions of the m1 receptors with Gi, Gq and G16 in BPH tissue and of the m1 and m3 receptors with Gi, Gq and G12 in PC3 and DU145 cells. Mitogen activated protein kinase (ERK) activity was seen in response to carbachol in PC3 and DU145 but not LnCaP cells. Finally, carbachol promoted cell proliferation in all three cell lines. Thus, there appears to be no consistent relationship between ERK activity, cell proliferation, and the subtype mediating the proliferative response, amongst these prostate cancer cell lines.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1995

Muscarinic receptor subtypes in normal, fetal, and gravid rabbit bladder, heart and uterus.

In the rabbit bladder, pregnancy has been shown to induce a significant decrease in both muscarinic receptor density and response to muscarinic stimulation. Neonatal rabbit bladders have a high muscarinic receptor density and contractile response to bethanechol stimulation. The bladders from 7 gravid rabbits, 7 age-matched virgin controls, and 32 fetal rabbits of 3 week gestation were studied. Compared to control tissue, filtration binding demonstrated receptor density to be 24.3% lower in gravid bladder dome, 41.2% lower in gravid bladder base, and 114.8% higher in fetal bladders. While total receptor density was not different from control in gravid heart, fetal hearts showed a 2.5 fold increased receptor density. There was also a 61% reduction in muscarinic receptor density in the gravid uterus. Immunoprecipitation assays using muscarinic receptor subtype specific antisera were used to measure the relative levels of m1, m2, m3 and m4 receptors. The m2 receptor was the predominant subtype in the bladder and uterus, and the only subtype detected in rabbit heart. The m3 receptor protein was also present, but in lower levels in the bladder and uterus. The m1 and m4 receptors were not detected in any of the tissues studied. Furthermore, the relative percent of each receptor did not statistically change for the gravid or fetal rabbit bladder, uterus, or heart, when compared to its control. Differences in the contractile response to cholinergic stimulation of the gravid bladder and uterus, and of the fetal bladder then, can be attributed to changes in muscarinic receptor density and not to changes in receptor subtype.

Animals↗
PubMed1995

Human prostate muscarinic receptor subtypes.

The alpha adrenergic receptor subtypes of the human prostate have been intensively investigated, while the muscarinic receptor subtypes and their function have yet to be determined in this tissue. [3H]-QNB binding to muscarinic receptors was performed on membrane homogenates of adenoma from six prostatectomy specimens resulting in an average total receptor density of 46 fMol/mg protein. Pirenzepine, hexahydrosiladifenidol, and para-fluoro-hexahydrosiladifenidol, drugs with high affinity for the M1 subtype, were significantly more potent inhibitors of [3H]-QNB binding than the M2 selective drug methoctramine. Immunoprecipitation studies were done using antisera raised to individual M1-M5 receptor subtypes. Approximately 75% of the solubilized receptors in the adenoma specimens were immunoprecipitated with the anti-M1 antibody, in contrast to 5% or less with antibodies against M2, M3 or M4 subtypes. These immunoprecipitation studies confirm the preponderance of the M1 subtype in prostate adenoma suggested by the high affinity pirenzepine binding. M1 receptors, when incubated with agonist, coimmunoprecipitated with the alpha subunits of the guanine nucleotide binding regulatory proteins Gi alpha, Gq/11 alpha and G16 alpha. Immunohistochemical staining with the anti-M1 antibody demonstrates the M1 receptor to be localized to the glandular epithelium. The human prostate is the first peripheral tissue in which a preponderance of the M1 subtype of muscarinic receptors has been demonstrated.

Adult↗
PubMed1995

Muscarinic acetylcholine receptor subtypes mediating urinary bladder contractility and coupling to GTP binding proteins.

Activation of muscarinic acetylcholine receptors is primarily responsible for urinary bladder emptying. Because multiple subtypes of muscarinic receptors exist, we wished to characterize those present in bladder and ultimately to attribute function to those that regulate bladder contractility, neurotransmitter release and perhaps other cholinergic functions in this tissue. Although the m2 and m3 subtypes could be immunoprecipitated after solubilization from human, rat, rabbit and guinea pig bladder membranes, the m1, m4 and m5 subtypes could not. The m2:m3 ratio was 9:1 in rat bladder but was only 3:1 in the other species examined. Immunoprecipitation of the m2 subtype correlated with the relative levels of high-affinity agonist binding sites measured by competition of carbachol for [3H]N-methylscopolamine binding or measured directly using [3H]oxotremorine-M. In the presence of agonist, but not antagonist, GTP binding proteins could be immunoprecipitated in concert with the m2 or m3 receptors using anti-receptor antibodies. These proteins were members of the Gi and Gq/11 subfamilies for both the m2 and the m3 receptor subtypes. In spite of the preponderance of the m2 receptor in all species studied, Schild analysis using somewhat selective antagonists showed that the pharmacologically defined m3 receptor mediated contractility in strips of rat and rabbit bladder. Thus acetylcholine activates bladder smooth muscle via the m3 receptor subtype, and subsequent contractility may be transduced by guanine nucleotide binding proteins such as the Gi and Gq/11 subfamilies.

Animals↗
PubMed1994

Bladder permeability in interstitial cystitis is similar to that of normal volunteers: direct measurement by transvesical absorption of 99mtechnetium-diethylenetriaminepentaacetic acid.

Bladder permeability was directly measured with the radionuclide used clinically for detecting vesicoureteral reflux (99mtechnetium-diethylenetriaminepentaacetic acid, 99mTc-DTPA) in 10 interstitial cystitis patients diagnosed according to National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases criterion and compared to 9 sex matched, symptom-free, normal volunteers. After functional bladder capacity was determined (capacity at which the patient demands fluid inflow to stop), the bladder was emptied and 5 mCi. 99mTc-DTPA in 10 ml. of saline were infused followed by normal saline to 80% of functional capacity. This was done to normalize the patients to the same low bladder pressure, since previous studies of rabbits indicated that bladder permeability is low and not significantly different at 20% and 60% of anesthetized bladder capacity (defined as the volume producing an intravesical pressure of 20 cm. water). Radioactivity of 1 ml. serum specimens taken at 0, 2, 15 and 30 minutes after radionuclide infusion was determined in a gamma counter, corrected for radioactive decay and converted to per cent of instilled dose per whole body based on blood volume estimated from body weight for each individual. There was considerable interindividual variability in the absorption between the patients and the volunteers. Analysis of variance of these data showed no statistically significant difference between the patients and controls at any time sampled. These results indicate that while some interstitial cystitis patients have a more permeable bladder than others, the same is true for normal, symptom-free volunteers. Thus, the concept of increased bladder permeability in interstitial cystitis is not supported by this direct measurement of bladder permeability.

Adult↗
PubMed1994

Identification and characterization of a high-affinity peripheral-type benzodiazepine receptor in rabbit urinary bladder.

The present study used radioligand binding and in vitro contractility experiments to identify and characterize a peripheral-type benzodiazepine receptor PBR in rabbit urinary bladder. [3H]PK11195 bound to bladder membranes with high-affinity and density (Kd = 5.2 nM., Bmax = 268 fmol./mg. protein), indicating the presence of a PBR. [3H]flunitrazepam bound with high-affinity and density (Kd = 1.2 nM., Bmax = 48 fmol./mg. protein). The rank order potency of various benzodiazepines and isoquinoline carboxamides in displacing the binding of [3H]PK11195 was Ro5-4864 > diazepam = flunitrazepam >> Ro15-1788 = clonazepam. Ro5-4864 and PK11195 inhibited nerve-evoked contractions in a concentration-dependent manner (IC50 = 42 microM. and 56 microM., respectively). Carbachol- and KCl-induced contractions were also inhibited by Ro5-4864 and PK11195. KCl-induced contractions were inhibited to a greater extent than carbachol-induced or field-stimulated contractions with all the drugs tested. Both Ro5-4864 and PK11195 significantly increased the ED50 for calcium-induced contractions following a cholinergic stimulus compared with control. These data demonstrate the presence of a PBR in urinary bladder capable of altering contractility in vitro through modulation of calcium activity.

Animals↗
PubMed1994

Current findings and future research avenues in the study of interstitial cystitis.

This article reports the progress made in the last several years of investigations into possible pathogenic processes that lead to the development of interstitial cystitis. The evidence presented here asserts that bladder mucin is functionally intact in interstitial cystitis and that the permeability of the afflicted patient's bladder is not greater than normal.

Animals↗