Frequency Electrical Field Stimulation Research Articles

Visualization of Exo- and Endocytosis of AMPA Receptors During Hippocampal Synaptic Plasticity Around Postsynaptic-Like Membrane Formed on Glass Surface.

Regulation of exo- and endocytosis of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor (AMPAR) plays a critical role in the expression of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD) at excitatory central synapses. Enhanced AMPAR exocytosis or endocytosis has been suggested to contribute to LTP or LTD, respectively. However, several unsettled fundamental questions have remained about AMPAR exo- and endocytosis in the basal condition and during synaptic plasticity: (1) Does the size of each exo- or endocytosis event, and/or do the frequencies of these events change during LTP or LTD? If they change, what are the time courses of the respective changes? (2) Where does the exo- or endocytosis preferentially occur in each condition: inside or in the vicinity of postsynaptic membrane, or in the extrasynaptic membrane? (3) Do different types of AMPAR, such as GluA1 homo-tetramer, GluA1/2 hetero-tetramer and GluA2/3 hetero-tetramer, show distinct exo- and endocytosis changes? To address these questions, we developed new methods to observe individual events of AMPAR exo- or endocytosis with a high signal to noise (SN) ratio in a culture preparation using total internal reflection fluorescence microscopy (TIRFM). In these studies, hippocampal neurons were cultured on a neurexin (NRX)-coated glass coverslip, which induced formation of postsynaptic-like membrane (PSLM) directly on the glass surface. Then, a super-ecliptic pHluorin (SEP)-tagged AMPAR subunit such as GluA1 (GluA1-SEP) was expressed in neurons and its fluorescence changes during LTP induced by high frequency electrical field stimulation were observed with TIRFM, which showed different time courses of exocytosis changes of GluA1-, GluA2-, or GluA3-SEP in and around PSLM. In addition, a new method to detect individual endocytosis events of AMPAR was developed by combining TIFRM observation of GluA-SEP around PSLM with a rapid extracellular pH exchange method using a U-tube. Recent results on exo- and endocytosis changes of GluA-SEP during N-methyl-D-aspartate (NMDA)-induced LTD suggested that suppression of AMPAR exocytosis rather than enhancement of AMPAR endocytosis primarily contributes to LTD expression, although the NMDA application transiently enhances clathrin-dependent endocytosis of GluA1-containing AMPAR.

Contractile Protein Expression and Phosphorylation and Contractility of Gastric Smooth Muscles from Obese Patients and Patients with Obesity and Diabetes.

Ingested food is received, mixed, and ground into chyme by distinct gastric motility patterns. Diabetes impairs gastric muscle function, but the mechanisms underlying diabetes-induced gastric muscle dysfunction are unknown. Here, we compared the expression and phosphorylation of Ca2+ sensitization and contractile proteins in human gastric muscles from obese nondiabetic and diabetic patients. We also compared the spontaneous phasic contractions and the contractile responses evoked by electrical field stimulation of cholinergic motor neurons. Fundus and antrum muscles were obtained from sleeve gastrectomies and were used in in vitro myobath contractile studies and for capillary electrophoresis and immunodetection of γ-actin, CPI-17, pT38-CPI-17, MYPT1, pT853-MYPT1, pT696-MYPT1, myosin light chain (MYL9), pS19-MYL9, myosin light chain kinase (MYLK), protein phosphatase-1δ (PP1δ), and Rho-associated kinase (ROCK2). In diabetic fundus muscles, MYLK, ROCK2, and PP1δ expression was unchanged; MYPT1 and CPI-17 expression was decreased; and the pT853/MYPT1 and pT38/CPI-17 ratios, but not the pT696/MYPT1 ratio, were increased. Although MYL9 expression was increased, the pS19/MYL9 ratio was unchanged in diabetic fundus muscles. In diabetic antrum muscles, MYLK and MYL9 expression was unchanged, but ROCK2, CPI-17, and PP1δ expression was decreased. The pT38/CPI-17 ratio was unchanged, while the pS19/MYL9, pT853/MYPT1, and pT696/MYPT1 ratios were decreased, consistent with the reduced ROCK2 expression. The frequencies of spontaneous phasic contractions from nondiabetic and diabetic gastric fundus and antrum muscles did not significantly differ from each other, regardless of age, sex, or diabetic status. The fold increases in the contractions of diabetic fundus and antrum muscles in response to increased frequencies of electrical field stimulation were significantly lower compared to nondiabetic fundus and antrum muscles. The altered contractile responses and the protein expression and phosphorylation in gastric muscles of obese patients with diabetes illustrate the importance of understanding how smooth muscle Ca2+ sensitization mechanisms contribute to gastric motility.

Effect of a Fructose Rich Diet in Vas Deferens Contractility in Rats

Objective: We aimed to investigate the effects of a Carbohydrate-rich diet on vas deferens contractility and histology. Materials and Methods: Twenty mature male rats were subjected into two groups. The control group received regular food and water and the study group received regular food and a carbohydrate-rich liquid diet mixture of 37–40% fructose, 30–36% sucrose and 27–30% glucose instead of water and prepared stock diet was diluted with 50% water. Animals were weighted and sacrificed after six weeks and the vas deferens has been evaluated with in vitro studies. Five rats from both the control and study groups, were separated into subgroups for dapoxetine administration 4 hours before they were sacrificed. Results: At the beginning of this study, the mean body weights were 228.6±6.7 g (range: 205-237) and 231.4±9.6 g (range: 202-243) in control and study groups, respectively. The mean body weights were 247.1±4.8 g (236-252) in the control group and 318.8±9.3 g (314-326) in the study group ( p 0.05). Responses to Adenosine Three Phosphate (ATP) were also not significant ( p >0.05). The contractile responses at the same frequencies of electrical field stimulation (EFS) were similar in both groups ( p >0.05). Histological examinations showed no abnormalities in either group. Conclusion: Carbohydrate-rich diet caused no increase in fibrotic activity, and pharmacological and histological properties remained stable, but caused significant weight gain over a short time period. Furthermore, dapoxetine had no effect on the contractility of the vas deferens.

A Novel Approach to Identify Genes Related to FGID

To investigate novel genes involved in functional gastrointestinal disorders, we disrupted a master regulator of mRNA splicing, the survival motor neuron (SMN) protein. SMN disruption leads to widespread changes in gene expression but has not been evaluated for effects in the gastrointestinal (GI) tract. Mice with a Nestin‐cre transgene were crossed to cre reporter mice to analyze expression in the enteric nervous system (ENS). Gene expression was detected in excitatory and sensory neurons, interneurons, and intraganglionic glia throughout the myenteric plexus but not muscle. Next, nestin‐cre mice were crossed to mice with a floxed Smn allele on a SMNΔ7 background (SMN2+/+;SMNΔ7+/+;SmnF7/‐) to produce Nestin‐F7 mice. In organ baths, SMN reduction disrupted colon longitudinal muscle contraction during high frequency electrical field stimulation (EFS). Muscle relaxation was consistently greater in control tissue. The addition of L‐NAME had no effect on differences between control and Nestin‐F7 responses. Tissues showed no morphological changes or cell loss. Gastric emptying and intestinal transit were significantly slower in Nestin‐F7 mice compared to controls. Nestin‐F7 mice had dry, infrequent stool that corresponded to reduced colonic motility in the bead latency test. Collectively, these data suggest that SMN reduction results in impaired GI function. Future studies will identify specific enteric cell types sensitive to SMN reduction and evaluate mRNA expression in those cells.

1026 PELVIC NERVE INJURY LEADS TO AUGMENTED ADRENERGIC CONSTRICTION AND FIBROSIS OF THE DISTAL VAGINA

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research (II)1 Apr 20131026 PELVIC NERVE INJURY LEADS TO AUGMENTED ADRENERGIC CONSTRICTION AND FIBROSIS OF THE DISTAL VAGINA Vinson M. Wang, Xiaopu Liu, Fabio Castiglione, Arthur L. Burnett, Petter Hedlund, Trinity J. Bivalacqua, and Johanna L. Hannan Vinson M. WangVinson M. Wang Baltimore, MD More articles by this author , Xiaopu LiuXiaopu Liu Baltimore, MD More articles by this author , Fabio CastiglioneFabio Castiglione Milan, Italy More articles by this author , Arthur L. BurnettArthur L. Burnett Baltimore, MD More articles by this author , Petter HedlundPetter Hedlund Lund, Sweden More articles by this author , Trinity J. BivalacquaTrinity J. Bivalacqua Baltimore, MD More articles by this author , and Johanna L. HannanJohanna L. Hannan Baltimore, MD More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.611AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Hysterectomy is the second most common surgery performed in women in the United States and frequently results in sexual dysfunction. Female sexual responses rely on innervations from the pelvic plexus which may become damaged during radical hysterectomy. The objective of this study is to characterize the physiological and morphological end organ changes that take place following bilateral pelvic nerve injury (BPNI) in female rats. METHODS Female Sprague-Dawley rats (12 wks) were divided into sham or BPNI in which nerves from the pelvic plexus innervating the vagina and bladder were bilaterally crushed. Animals were examined 7, 14, 30 and 60 days following BPNI. Distal vaginal strips were mounted in a myograph and contractile/relaxation responses to increasing concentrations of norepinephrine (NE) or frequencies of electrical field stimulation (EFS) were measured in the presence or absence of nitric oxide synthase (NOS) inhibitor L-NAME. Rho-kinase (ROCK1, ROCK2) and RhoA gene expression were also assessed by PCR. Vaginal segments were formalin-fixed and stained with MassonÆs trichrome to assess smooth muscle and collagen content. RESULTS There is a significant increase in maximal vaginal contraction to NE at 7 and 14 days following BPNI (Sham: 8.1mN; 7d: 14.4mN; 14d: 12.2mN; p<0.05). At 30 and 60 days following injury contractions to NE had returned to sham levels. EFS mediated contractions were only slightly increased after BPNI. The increases in EFS contraction were exacerbated in the presence of L-NAME indicating that NOS may be compromised following nerve injury. Relaxation to non-adrenergic non-cholinergic EFS stimulation was significantly decreased 7 and 14 days after BPNI (Sham: 24.3%; 7d: 15.3%; 14d:11.1% relaxation, p<0.05) and returned to sham levels by 30 days. Gene expression of ROCK1 and ROCK2 was significantly increased 14 days after BPNI (p<0.05) and itÆs activator RhoA was also significantly increased at 7 and 14 days compared to sham (p<0.05). Additionally, 14 days after BPNI there was an increase in collagen within the vaginal wall and a decrease in vaginal epithelium. CONCLUSIONS These results demonstrate that pelvic nerve injury leads to increased adrenergic, rho-kinase mediated tone, impaired relaxation and structural changes to the vagina. These findings parallel the phenotype demonstrated in the male cavernous nerve injury model and provide us with a female counterpart to study the mechanisms of neuropraxia-induced female sexual dysfunction. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e420-e421 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Vinson M. Wang Baltimore, MD More articles by this author Xiaopu Liu Baltimore, MD More articles by this author Fabio Castiglione Milan, Italy More articles by this author Arthur L. Burnett Baltimore, MD More articles by this author Petter Hedlund Lund, Sweden More articles by this author Trinity J. Bivalacqua Baltimore, MD More articles by this author Johanna L. Hannan Baltimore, MD More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

120 CHOLINERGIC MEDIATED DETRUSOR DYSFUNCTION IN A FEMALE RAT MODEL OF PELVIC NEUROPRAXIA

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Basic Research (II)1 Apr 2013120 CHOLINERGIC MEDIATED DETRUSOR DYSFUNCTION IN A FEMALE RAT MODEL OF PELVIC NEUROPRAXIA Johanna L. Hannan, Vinson M. Wang, Xiaopu Liu, Fabio Castiglione, Arthur L. Burnett, Petter Hedlund, and Trinity J. Bivalacqua Johanna L. HannanJohanna L. Hannan Baltimore, MD More articles by this author , Vinson M. WangVinson M. Wang Baltimore, MD More articles by this author , Xiaopu LiuXiaopu Liu Baltimore, MD More articles by this author , Fabio CastiglioneFabio Castiglione Milan, Italy More articles by this author , Arthur L. BurnettArthur L. Burnett Baltimore, MD More articles by this author , Petter HedlundPetter Hedlund Lund, Sweden More articles by this author , and Trinity J. BivalacquaTrinity J. Bivalacqua Baltimore, MD More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1499AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urogynaecological surgeries for cervical or colorectal cancer often result in damage to the neurovascular bundle and pelvic plexus leading to voiding dysfunction. Although surgical techniques have improved mortality rates in women, the prevalence of voiding dysfunctions severely impacts their quality of life. The objective of this study is to characterize the physiological and molecular changes in the bladder that take place following bilateral pelvic nerve injury (BPNI) in female rats. METHODS Female Sprague-Dawley rats (12 wks, n=40) were divided into sham or BPNI in which nerves from the pelvic plexus innervating bladder were bilaterally crushed. Animals were examined 7, 14, 30 and 60 days following BPNI. The bladder was excised and weighed. Detrusor strips were mounted in a myograph and contractile responses to increasing concentrations of carbachol or frequencies of electrical field stimulation (EFS) were assessed. Gene expression of muscarinic type 2 (MR2) and 3 (MR3) receptors was also assessed by RTPCR. RESULTS There was a significant increase in the bladder weight at 7 and 14 days following nerve injury (Sham: 3.1%; 7d: 3.9%; 14d: 4.3% of body weight, p<0.05). Maximal carbachol-mediated contraction was decreased following nerve injury at all time points (Sham: 17.9mN; 7d: 12.3mN; 14d: 12.8mN; 30d: 13.4mN; 60d: 14.8mN; p<0.05). Furthermore, the EC50 was markedly increased at 14 and 30 days after BPNI (Sham: 7.0 pD2; 14d: 6.0 pD2; 30d: 6.2 pD2, p<0.05) indicating that a higher concentration of carbachol was required to achieve 50% of maximal contraction. By 60 days following BPNI, maximal contraction and EC50 to carbachol had returned to sham levels. EFS mediated contraction was also significantly reduced after nerve injury when stimulate at lower frequencies (<8Hz) but all groups achieved the same maximal contraction at high frequency stimulation (32Hz). Gene expression of MR2 was significantly lower 7 days following nerve injury (Sham: 0.91; 7d: 0.34 MR2 relative expression, p<0.05); however gene expression of MR3 was unchanged 7days following BPNI. CONCLUSIONS These results demonstrate that pelvic nerve injury leads to a temporal increase in bladder weight and decreased cholinergic mediated contractions and MR2 gene expression. These data suggest that this animal model can be used to study the mechanism of neuropraxia-induced voiding dysfunction in female rats. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e48 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Johanna L. Hannan Baltimore, MD More articles by this author Vinson M. Wang Baltimore, MD More articles by this author Xiaopu Liu Baltimore, MD More articles by this author Fabio Castiglione Milan, Italy More articles by this author Arthur L. Burnett Baltimore, MD More articles by this author Petter Hedlund Lund, Sweden More articles by this author Trinity J. Bivalacqua Baltimore, MD More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Rate and irregularity of electrical activation during atrial fibrillation affect myocardial NGF expression via different signalling routes

An irregular ventricular response during atrial fibrillation (AF) has been shown to mediate an increase in sympathetic nerve activity in human subjects. The molecular mechanisms remain unclear. This study aimed to investigate the impact of rate and irregularity on nerve growth factor (NGF) expression in cardiomyocytes, since NGF is known to be the main contributor to cardiac sympathetic innervation density. Cell cultures of neonatal rat ventricular myocytes were electrically stimulated for 48h with increasing rates (0, 5 and 50Hz) and irregularity (standard deviation (SD)=5%, 25% and 50% of mean cycle length). Furthermore, we analyzed the calcineurin-NFAT and the endothelin-1 signalling pathways as possible contributors to NGF regulation during arrhythmic stimulation. We found that the increase of NGF expression reached its maximum at the irregularity of 25% SD by 5Hz (NGF: 5Hz 0% SD=1 vs. 5Hz 25% SD=1.57, P<0.05). Specific blockade of the ET-A receptor by BQ123 could abolish this NGF increase (NGF: 5Hz 25% SD+BQ123=0.66, P<0.05). High frequency electrical field stimulation (HFES) with 50Hz decreased the NGF expression in a significant manner (NGF: 50Hz=0.55, P<0.05). Inhibition of calcineurin-NFAT signalling with cyclosporine-A or 11R-VIVIT abolished the HFES induced NGF down-regulation (NGF: 50Hz+CsA=1.14, P<0.05). In summary, this study reveals different signalling routes of NGF expression in cardiomyocytes exposed to increasing rates and irregularity. Whether this translates into different degrees of NGF expression and possibly neural sympathetic growth in various forms of ventricular rate control during AF remains to be elucidated in further studies.

Sympathetic Neurons Express and Secrete MMP-2 and MT1-MMP to Control Nerve Sprouting via Pro-NGF Conversion

Recently, we have shown that high frequency electrical field stimulation (HFES) of sympathetic neurons (SN) induces nerve sprouting by up-regulation of nerve growth factor (NGF) which targets the tyrosine kinase A receptor (TrkA) in an autocrine/paracrine manner. There is increasing evidence that matrix metalloproteinase-2 (MMP-2) is not only involved in extracellular matrix (ECM) turnover but may also exert beneficial effects during neuronal growth. Therefore, this study aimed to investigate the regulation and function of MMP-2 and its major activator membrane type 1-matrix metalloproteinase (MT1-MMP) as well its inhibitor TIMP-1 in SN under conditions of HFES. Moreover, we analyzed molecular mechanisms of the beneficial effect of losartan, an angiotensin II type I receptor (AT-1)blocker on HFES-induced nerve sprouting. Cell cultures of SN from the superior cervical ganglia (SCG) of neonatal rats were electrically stimulated for 48 h with a frequency of 5 or 50 Hz. HFES increased MMP-2 and MT1-MMP mRNA and protein expression, whereas TIMP-1 expression remained unchanged. Under conditions of HFES, we observed a shift from pro- to active-MMP-2 indicating an increase in MMP-2 enzyme activity. Specific pharmacological MMP-2 inhibition contributed to an increase in pro-NGF amount in the cell culture supernatant and significantly reduced HFES-induced neurite outgrowth. Losartan abolished HFES-induced nerve sprouting in a significant manner by preventing HFES-induced NGF, MMP-2, and MT1-MMP up-regulation. In summary, specific MMP-2 blockade prevents sympathetic nerve sprouting (SNS) by inhibition of pro-NGF conversion while losartan abolishes HFES-induced SNS by reducing total NGF, MMP-2 and MT1-MMP expression.

Electrical stimulation of sympathetic neurons induces autocrine/paracrine effects of NGF mediated by TrkA

Neuronal remodeling with increased sympathetic innervation density has been implicated in the pathogenesis of atrial fibrillation (AF). Recently, increased transcardiac nerve growth factor (NGF) levels were observed in a canine model of AF. Whether atrial myocytes or cardiac sympathetic neurons are the source of neurotrophins, and whether NGF is the main neurotrophic factor contributing to sympathetic nerve sprouting (SNS) in AF still remains unclear. Therefore, neonatal rat atrial myocytes were cultured under conditions of high frequency electrical field stimulation (HFES) to mimic rapid atrial depolarization. Likewise, sympathetic neurons from the superior cervical ganglia of neonatal rats were exposed to HFES to simulate the physiological effect of sympathetic stimulation. Real-time PCR, ELISA and Western blots were performed to analyze the expression pattern of NGF and neurotrophin-3 (NT-3). Baseline NGF and NT-3 content was 3-fold higher in sympathetic neurons than in atrial myocytes (relative NGF protein expression: 1±0.0 vs. 0.37±0.11, all n=5, p<0.05). HFES of sympathetic neurons induced a frequency dependent NGF and NT-3 gene and protein up-regulation (relative NGF protein expression: 0Hz=1±0.0 vs. 5Hz=1.13±0.19 vs. 50Hz=1.77±0.08, all n=5, 0Hz/5Hz vs. 50Hz p<0.05), with a subsequent increase of growth associated protein 43 (GAP-43) expression and morphological SNS. Moreover, HFES of sympathetic neurons increased the tyrosine kinase A (TrkA) receptor expression. HFES induced neurotrophic effects could be abolished by lidocaine, TrkA blockade or NGF neutralizing antibodies, while NT-3 neutralizing antibodies had no significant effect on SNS. In neonatal rat atrial myocytes, HFES resulted in myocyte hypertrophy accompanied by an increase in NT-3 and a decrease in NGF expression. In summary, this study provides evidence that high-rate electrical stimulation of sympathetic neurons mediates nerve sprouting by an increase in NGF expression that targets the TrkA receptor in an autocrine/paracrine manner.

Effect of Botulinum Toxin A on the Autonomic Nervous System of the Rat Lower Urinary Tract

The magnitude and duration of the effects of botulinum toxin A on acetylcholine (ACh) and norepinephrine release from the bladder and urethra of rats were measured using a radiochemical method. Saline (sham treatment) or botulinum toxin A was injected into the bladder (50 microl.) or urethra (30 microl.) in separate groups of animals. The release of 3H-norepinephrine or 14C-choline was measured at 2 time points after injection (5 or 30 days). The fractional release of ACh in botulinum toxin A treated animals was significantly inhibited at higher frequencies of electrical field stimulation (20 Hz.) but not at lower frequencies (2 Hz.) 5 days after injection. However, ACh release recovered to sham injected values 30 days after toxin injection. No significant differences in the fractional release of norepinephrine from sham injected or botulinum toxin A bladders were observed. In contrast, norepinephrine release from the urethra was inhibited by botulinum toxin A for at least 30 days after injection. Similar to its effect on transmitter release in the bladder, botulinum toxin A inhibited norepinephrine release in the urethra at high (20 Hz.) but not at low (4 Hz.) electrical stimulation frequencies. These data indicate that the clinical effects of botulinum toxin A on the lower urinary tract may vary depending on the site of injection and level of nerve activity.

Idiopathic chronic constipation: tachykinins as cotransmitters in colonic contraction.

Tachykinins (TKs) have been shown to be involved in the excitatory enteric motor pathway. This study aimed to examine the direct and nerve-mediated effect of specific NK1, NK2 and NK3 receptor agonists and antagonists in colonic preparations from control subjects and patients with idiopathic chronic constipation (ICC). Cumulative concentrations of Sar9Met(O2)11 substance P (selective NK1 receptor agonist), [Ala5,beta-Ala8]-neurokinin A (4-10) (selective NK2 receptor agonist) and [MePhe7]-neurokinin B (selective NK3 receptor agonist) were tested on colonic circular muscle strips to evaluate the direct drug effects. In addition, in the presence of atropine, the role of TKs in the off-contraction that follows the typical inhibitory response evoked by low frequencies of electrical field stimulation (0.5--10 Hz, 20 V, 1 ms pulse trains lasting 1 min) was investigated. In control preparations, the rank order of potency was: NK2 receptor-selective agonist > NK3 receptor-selective agonist > NK1 receptor-selective agonist. The off-contraction was found to be reduced by about 30--40% in colonic circular muscle from ICC patients with respect to controls. Incubation with the NK1 receptor agonist did not modify the off-contraction measurements in either control or ICC preparations. Conversely, both NK2 and NK3 receptor agonists significantly (P < 0.01) increased the off-contraction in ICC preparations only. This increased response was fully antagonized by MEN-10627, a NK2 and NK3 receptor antagonist depending on the dose. We may conclude that ICC is hyporesponsive to TKs and that the contractile reflex to distension is greatly reduced in ICC disease, but can be restored by incubation with NK2 and NK3 receptor agonists.

Effects of erythromycin on human colonic circular muscle in idiopathic chronic constipation.

Erythromycin has been shown to have profound prokinetic effects on the gastrointestinal tract of humans and animals, probably through its action on endogenous motilin receptors. The purpose of this study was to determine both the direct and indirect effects ('off contraction') of erythromycin and motilin on ex vivo circular muscle strips of the distal colon from patients with or without idiopathic chronic constipation (ICC). Cumulative concentrations of erythromycin (1-20 microM) and motilin (0.05-1 microM) were tested in both control and ICC preparations in order to evaluate the direct drugs effect. A range doses of both erythromycin (0.5-10 microM) and motilin (0.05-0.5 microM) were tested on their ability to affect the off-contraction that follows the typical inhibitory response evoked by low frequencies of Electrical Field Stimulation (EFS) (1-5 Hz, 20 V, 1 msec pulse trains lasting 1 min). The direct effect of both erythromycin and motilin was a slight increase (less than 10% of the maximal ACh-induced contraction) in the basal tension, with no dose-response relationship. The off-contraction, evoked by EFS, was not affected by drugs pretreament in control preparations. Conversely, in ICC preparations both drugs significantly increased the off-contraction (about 30%). Erythromycin causes mainly an indirect contractile effect in circular muscle strips from ICC patients. This effect may be related to the activation of inhibitory neuronal motilin receptors. This activation might potentiate NANC relaxation, proportionally increasing the circumferential reflex contraction that follows the EFS-induced relaxation.

Neuropeptide Y accounts for sympathetic vasoconstriction in guinea-pig vena cava: evidence using BIBP 3226 and 3435

The ability of the novel, non-peptide, neuropeptide Y Y 1 receptor antagonist, BIBP 3226 (( R)- N 2-(diphenylacetyl)- N-[(4-hydroxyphenyl)methyl]-argininamide), to antagonize neuropeptide Y- and sympathetic-mediated vasoconstriction was examined in isolated segments of the thoracic vena cava of guinea-pigs. Increasing concentrations (10 −9–10 −6 M) of BIBP 3226 caused a parallel and rightward shift in the neuropeptide Y dose-response curve but did not significantly change the effect of noradrenaline. The calculated pA 2 value for BIBP 3226 was 8.0 ± 0.08, a value fully compatible with the reported affinity at rodent and human neuronal Y 1 receptors. BIBP 3226 (10 −6 M) also readily reversed the established vasocontraction induced by neuropeptide Y. BIBP 3226 (10 −6 M) markedly inhibited the slow long-lasting contraction evoked by high frequency electrical field stimulation, leaving a rapid component which was abolished by phentolamine. Its enantiomer, BIBP 3435 (( S)- N 2-(diphenylacetyl)- N-[(4-hydroxyphenyl)methyl]-argininamide), which exerts a much weaker action on neuropeptide Y Y 1 receptors, had no such inhibitory effect. In propranolol-pretreated vessels, the vasoconstriction evoked by nerve stimulation was enhanced; then BIBP 3226 inhibited the peak response by 44%, and the integrated contractile effect by 90%. We conclude that BIBP 3226 is a potent and competitive antagonist of neuropeptide Y Y 1 receptor-mediated vasoconstriction in guinea-pig vena cava and that endogenous neuropeptide Y acting on the neuropeptide Y Y 1 receptor is likely to account for the long-lasting component of the sympathetic vasoconstriction in response to high-frequency stimulation in this vessel.

In‐vitro contractile response of the rabbit corpus cavernosa to field stimulation and autonomic agonists and antagonists: A qualitative study

AbstractField stimulation of rabbit corporus cavernosum tissue strips can result in relaxation, contraction, or a biphasic response depending on the frequency and the power utilized. In this study we characterized the autonomic components of this response by exposing corporal tissue strips to a variety of autonomic agonists and antagonists including phentolamine, isoproterenol, methoxamine, propranolol, bethanechol, atropine, and ATP. Low frequency electrical field stimulation produced a bi‐phasic response characterized by an initial relaxation followed by a contraction. High frequencies (≥ 32 Hz) produced contraction only. All responses were abolished by tetrodotoxin, indicating that both the contractile and relaxant responses to field stimulation are mediated by complex neural mechanisms. The initial relaxation response involves a combination of mediators which include musca‐rinic cholinergic stimulation, purinergic, beta‐adrenergic, and non‐adrenergic, non‐cholinergic (NANC) stimulation. The non‐cholinergic contribution to corporal smooth muscle relaxation appeared to be approximately equal in significance to the cholinergically mediated relaxation. Beta adrenergic stimulation mediated a direct relaxation of the corporal smooth muscle. The contractile portion of the bi‐phasic response was mediated by alpha‐adrenergic stimulation. Additionally we have noted a rebound contraction following termination of field stimulation at all frequencies that is not affected by adrenergic or cholinergic blockade and may reflect the field‐stimulated release of an endogenous smooth muscle contractile factor.

Serotonin release varies with brain tryptophan levels

This study examines directly the effects on serotonin release of varying brain tryptophan levels within the physiologic range. It also addresses possible interactions between tryptophan availability and frequency of membrane depolarization in controlling serotonin release. We demonstrate that reducing tryptophan levels in rat hypothalamic slices (by superfusing them with medium supplemented with 100 μM leucine) decreases tissue serotonin levels as well as both spontaneous and electrically-evoked serotonin release. Conversely, elevating tissue tryptophan levels (by superfusing slices with medium supplemented with 2 μM tryptophan) increases both tissue serotonin levels and serotonin release. Serotonin release was found to be affected independently by tryptophan availability and frequency of electrical field-stimulation (1–5 Hz), since increasing both variables produced nearly additive increases in release. These observations demonstrate for the first time that both precursor-dependent elevations and reductions in brain serotonin levels produce proportionate changes in serotonin release, and that the magnitude of the tryptophan effect is unrelated to neuronal firing frequency. The data support the hypothesis that serotonin release is proportionate to intracellular serotonin levels.

In vivo exposure to hyperoxia increases airway responsiveness in rats. Demonstration in vivo and in vitro.

Studies regarding O2-induced lung injury have concentrated on damage to alveolar structures and pulmonary vasculature without consideration of alterations that may be occurring in airways. This study was undertaken to determine the effects of in vivo hyperoxic exposure on airway responses to excitatory stimuli in intact, anesthetized rats and in intrapulmonary bronchi isolated from hyperoxia-exposed rats. Using lung conductance (G1) as an index of bronchoconstriction, intravenously administered 5-hydroxytryptamine (5HT) elicited greater bronchoconstrictor responses in anesthetized, mechanically ventilated rats that had been exposed to 85% O2 for 7 days rather than to air. Further, airways of hyperoxia-exposed rats were more sensitive to the effects of intravenously administered 5HT as evidenced by the lower log dose of 5HT required to decrease G1 30%. Cylindrical segments of intrapulmonary bronchi isolated from hyperoxia-exposed rats were more responsive to the contractile effects of 5HT and electrical field stimulation. However, no differences in responsiveness to bethanechol or KCl were observed between the two groups. The log concentration of 5HT and the log frequency of electrical field stimulation that elicited half-maximal responses were smaller in bronchi isolated from hyperoxia-exposed animals, indicating an increase in sensitivity of the airways to these stimuli. These results suggest that prolonged exposure to greater than ambient levels of O2 can alter airway function; however, the mechanism responsible for these changes remains to be determined.

Selective depletion of the acetylcholine and vasoactive intestinal polypeptide of the guinea-pig myenteric plexus by differential mobilization of distinct transmitter pools.

The effect of electrical field stimulation on the release of acetylcholine (ACh) and vasoactive intestinal polypeptide (VIP) from superfused strips of myenteric plexus-longitudinal muscle (MPLM) of guinea-pig ileum and on the transmitter content of the tissue was investigated at different frequencies and in the presence and absence of choline hemicholinium-3 and colchicine. Low frequency electrical field stimulation released ACh by more than 4 times the basal release; the simultaneously detected VIP secretion was increased only slightly above the resting level. During high frequency stimulation (50 Hz) the release of VIP was greatly increased (to 5 times the resting release) whereas the release of ACh increased to only 150% of the basal output. When choline was present, the ACh content of the tissue itself was not altered by electrical stimulation indicating a rate of synthesis sufficient to maintain release. It was reduced in a frequency-dependent manner in the absence of exogenous choline or in the presence of 10 microM hemicholinium-3 (an inhibitor of choline uptake) by up to 54% of the original content. A similar but even larger reduction took place in the amount of ACh released. Neither the secretion of VIP nor the tissue VIP content was altered by these treatments. Long-lasting (greater than 60 min) high-frequency (50 Hz) stimulation resulted in the depletion of the VIP pool (by 25%) while the ACh content remained unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

Picric acid functions as a releaser of [14C]acetylcholine in isolated ileal synaptosomal preparations of guinea-pig.

The effect of picric acid on the release of [14C]acetylcholine has been investigated in isolated ileal synaptosomes of guinea-pig. Nicotine, high K-depolarization (50 mM KCl) and electrical field stimulation were employed to characterize the specificity of picric acid. Picric acid induced the release of labelled acetylcholine in a dose-dependent manner and this action was negated by the removal of calcium ions from the bathing medium. Tetrodotoxin (0.1 microM) abolished the actions of picric acid, nicotine or electrical field stimulation (0.1 Hz). It reduced but did not totally suppress the effect of high K-depolarization. Agents capable of affecting the content of cyclic AMP, such as forskolin and alloxan, modified the effects of picric acid or nicotine but did not influence the effects of high K-depolarization or electrical field stimulation. Indomethacin, at a concentration (1 microM) effective in inhibiting the synthesis of prostaglandins, reduced the release of acetylcholine evoked by picric acid or nicotine, but did not affect the responses to high K-depolarization or electrical field stimulation. [3H]5-hydroxytryptamine was also released by high K-depolarization at a concentration sufficient to induce the release of acetylcholine. Similar results were obtained when the frequency of electrical field stimulation was raised to 10 Hz. However, picric acid did not initiate the release of 5-hydroxytryptamine. These results suggest that picric acid functions as a releaser of acetylcholine through a mechanism different from that of other stimulants.

Adenosine and dipyridamole: actions and interactions on the contractile response of guinea-pig ileum to high frequency electrical field stimulation.

The action of adenosine on the myenteric plexus-longitudinal muscle strips from guinea-pig ileum to high frequency electrical field stimulation (10 Hz) was investigated. Electrically induced contractions were reduced markedly by tetrodotoxin (0.2 microM) and atropine (1 microM), and partially by noradrenaline (3 microM) and morphine (3 microM). Adenosine, adenosine 5'-monophosphate (AMP) and adenosine triphosphate (ATP) produced a concentration-dependent inhibition of the high frequency contractions over the range of 0.1-100 microM, the most potent being adenosine. The concentration-response curve for adenosine was significantly shifted to the left by dipyridamole (10 nM), while dipyridamole at higher concentrations (30 nM-10 microM), depressed the contraction markedly by itself. Dipyridamole decreased [3H]-adenosine uptake into strips of ileum in a concentration-dependent manner. There was a significant correlationship between the reduction of adenosine uptake and the inhibition of the contraction induced by dipyridamole (r = 0.970). In strips desensitized to adenosine or treated with adenosine deaminase, the inhibitory effect of dipyridamole was significantly reduced. The present investigation revealed that adenosine depressed responses of guinea-pig ileum to high frequency electrical stimulation and suggested that the inhibitory effect of dipyridamole may be closely associated with the behaviour of endogenous adenosine or related compounds.