High concentrations of landiolol,a β<Subscript>1</Subscript>-adrenoceptor antagonist,stimulate smooth muscle contraction of the rat trachea through the Rho-kinase pathway

Purpose Gradually progressing contraction of airway smooth muscle is suggested to be due to the Rho-kinase signaling pathway. In our preliminary study in rat tracheas, landiolol, a β₁-adrenoceptor antagonist, at high doses caused gradually progressing contraction, and this contraction reached a plateau after 20 min. Therefore, this study was carried out to clarify whether landiolol could stimulate the Rho-kinase pathway or the phosphatidylinositol (PI) response in the rat trachea. Methods Seventy-eight male Wistar rats weighing 250–350 g were used for the experiments. Their tracheas were cut into 3-mm-wide ring segments or 1-mm-wide slices. Measurements of isometric tension and [³H] inositol monophosphate (IP₁) production were conducted, using these tracheal rings or slices. Data values are expressed as means ± SD, and statistical significance (P < 0.05) was determined using analysis of variance (ANOVA). Results Landiolol (700 μM)-induced contraction was completely inhibited by fasudil at 30 μM, while the landiolol-induced contraction was not inhibited by 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP), ketanserin, or nicardipine. Landiolol did not stimulate IP₁ production. Conclusion These results suggest that high concentrations of landiolol could cause airway smooth muscle contraction through the Rho-kinase pathway, but not through the PI response coupled with muscarinic M₃ receptors, 5-HT receptors or the activation of L-type Ca²⁺ channels.     

Effects of ketamine on neostigmine-induced contractile and phosphatidylinositol responses of the rat trachea

Purpose. Neostigmine causes airway smooth muscle contraction through the direct stimulation of muscarinic receptors and the activation of phosphatidylinositol (PI) responses. Ketamine attenuates airway smooth muscle contraction. It is not clear whether ketamine attenuates neostigmine-induced airway smooth muscle contraction by inhibiting the PI response. This study was designed to examine the effects of ketamine on neostigmine-induced contractile and PI responses of the rat trachea. Methods. Thirty male Wistar rats weighing 250–350 g were used. In the experiment on the contractile response, active contraction was induced with 1 μM neostigmine in the presence or absence of ketamine. In the experiment on the phosphatidylinositol response, the trachea slices were incubated with [³H]myo-inositol, 1 μM neostigmine, or 100 μM aluminum fluoride, and ketamine. The formation of [³H]inositol monophosphate (IP₁), a degradation product of the phosphatidylinositol response, was measured with a liquid scintillation counter. Statistical significance (P < 0.05) was determined by analysis of variance. Results. Neostigmine 1 μM caused tracheal ring contraction. This contraction was attenuated by ketamine dose-dependently and reached resting tension at 100 μM. Neostigmine- and aluminum fluoride-induced IP₁ accumulation was also attenuated by ketamine. Conclusion. The results suggest that ketamine attenuates neostigmine-induced contractile responses, at least in part, through the inhibition of phospholipase C coupled with G protein in the PI response. Received: December 12, 2002 / Accepted: February 8, 2003 Acknowledgments. This study was supported in part by Grant-in-Aid for Scientific Research C, No. 10671421, from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. Address correspondence to: M. Saito     

Effects of vasopressors on contractile and phosphatidylinositol responses of rat trachea

Purpose. Vasopressors, such as dopamine (DA), norepinephrine (NE), and phenylephrine (Phe), are commonly used during anesthesia to increase blood pressure through α₁-adrenoceptors. The present study was designed to examine the effects of DA, NE, and Phe on the contractile and phosphatidylinositol (PI) responses of the rat trachea induced by a muscarinic agonist, carbachol (CCh). Methods. A rat tracheal ring was suspended between two stainless-steel hooks in Krebs-Henseleit (K-H) solution. Contraction was induced with 0.55 μM CCh, and 30 min later DA, NE, or Phe was added. The tracheal slices were incubated in K-H solution containing LiCl, ³[H]myo-inositol, and CCh in the presence or absence of DA, NE, or Phe. The ³[H]inositol monophosphate (IP₁) formed was measured. Results. CCh caused tracheal ring contraction. NE attenuated CCh-induced contraction at a dose of 1 μM or greater and had a maximal effect at 3 μM. DA and Phe did not affect CCh-induced contraction. CCh-induced IP₁ accumulation was potentiated significantly by NE and Phe, but not by DA. Conclusion. Although NE and Phe potentiated CCh-induced IP₁ accumulation, they could not potentiate CCh-induced contraction, suggesting that in clinical settings, vasopressors such as NE, DA, and Phe might be safely used in patients with asthma. Received: February 25, 2002 / Accepted: July 2, 2002 Acknowledgments. This study was supported in part by a Grant-in-Aid for Scientific Research C (no. 10671421), from the Ministry of Education, Science, and Culture, Japan. Address correspondence to: O. Shibata     

Effects of ATP-sensitive potassium channel openers on the contractile and phosphatidylinositol responses of the rat trachea

Purpose. Although ATP-sensitive potassium channel openers suppress airway smooth muscle contraction, their potencies are different and the mechanisms involved are not fully understood. We examined the effects of cromakalim and Y-26763, a novel ATP-sensitive potassium channel opener, on the contractile and phosphatidylinositol responses of the rat trachea. Methods. Thirty-six male Wistar rats, weighing 250–350 g, were used. In the experiment on contractile response, active contraction was induced with 0.55 μM carbachol in the presence or absence of cromakalim or Y-26763. In the experiment on phosphatidylinositol response, the tracheal slices were incubated with [³H]myo-inositol, 0.55 μM carbachol, and cromakalim or Y-26763, and the formation of [³H]inositol monophosphate (IP₁), a degradation product of phosphatidylinositol response, was measured with a liquid scintillation counter. Statistical significance (P < 0.05) was determined by analysis of variance (ANOVA). Results. Carbachol-induced tension was attenuated by both cromakalim and Y-26763, the latter displaying significantly greater potency. Carbachol-induced IP₁ accumulation was influenced neither by cromakalim nor by Y-26763. Conclusion. Both cromakalim and Y-26763 have effects on airway smooth muscle relaxation. Carbachol-induced IP₁ accumulation was influenced neither by cromakalim nor by Y-26763, suggesting that phosphatidylinositol response may not be a common pathway for the effect of ATP-sensitive potassium channel openers. Received: January 15, 2002 / Accepted: June 14, 2002 Acknowledgment. This study was supported in part by Grant-in-Aid for Scientific Research C, no. 10671421, from the Ministry of Education, Science, and Culture, Japan. Address correspondence to: O. Shibata     

Effects of inositol trisphosphate on calcium mobilization in bone cells

Summary The effect of inositol 1,4,5 trisphosphate (IP₃) on calcium mobilization was studied in human osteosarcoma lines, Saos-2 and G292, as well as isolated rat osteoblastic and osteoclastic cells. Cells were permeabilized with saponin and calcium mobilization was studied with the fluorescent dye, fura-2 in a recording spectrofluorometer. IP₃ (10 μM) increased calcium release in all cell types studied. The effect was dependent on ATP and occurred in the presence of mitochondrial inhibitors. The effect was not seen with inositol 1-phosphate (IP) or inositol 1,4-diphosphate (IP₂). Inositol 1,3,4,5 tetrakisphosphate (IP₄) appeared to elicit a decrease in the calcium released. Depletion of the intracellular pool with the calcium ionophore, ionomycin, as well as incubation with the inhibitor of intracellular calcium mobilization, TMB-8, obliterated the IP₃ effect. The results are consistent with the hypothesis that increases in IP₃ can cause a rapid elevation of bone cell cytosolic calcium.     

Histoculture drug response assay for gefitinib in non-small-cell lung cancer

Objective  There are many predictive factors for gefitinib sensitivity, including epidermal growth factor receptor (EGFR) gene mutation, EGFR copy number, and k-ras mutation. To investigate all of them is too expensive. We evaluated the chemosensitivity for gefitinib in non-small-cell lung cancer (NSCLC) using a histoculture drug response assay (HDRA). Methods  Surgically resected fresh tumor specimens from 22 patients with NSCLC were used. There were 13 male and 9 female patients, ranging in age from 49 to 84 (average 70) years old. Sixteen patients (73%) were smokers. Sixteen adenocarcinomas, four squamous cell carcinomas, and two other histological types were included. Small pieces of viable cancer tissue were placed on the collagen gel and then cultured for 7 days in the presence of gefitinib. Results  The HDRA was successful in all specimens. A dose-response relation was observed between inhibition rates and gefitinib concentration (p = 0.016). The inhibition rate at 20 μg/ml (IR₂₀) in adenocarcinoma without smoking (39.2% ± 35.1%, n = 6) was higher than that with smoking (2.2% ± 5.0%, n = 10, P = 0.001) and that of nonadenocarcinoma (16.9% ± 23.6%, n = 6, P = 0.09). Gene mutation analysis was performed in two of three adenocarcinomas without smoking, which showed especially high IR₂₀ values, and sensitizing mutations were observed in these specimens. A cutoff inhibition rate of approximately 40%–50% appeared to be suitable for a concentration of 20 μg/ml. Conclusion  HDRA appears to be applicable for evaluating sensitivity to gefitinib in NSCLC. It provides a convenient method for predicting the response to gefitinib in patients with NSCLC whose fresh tumor specimens are available.     

In vitro study of rat prostate 5α-reductase activity and its inhibition

A simple and rapid method of measuring 5α-reductase (5α-R) activity and of determining the kinetic parameters (K_M and V_max) of the enzyme is described. The 5α-R activity in the homogenate of the prostate of Wistar rats aged 8–12 weeks was established, and the effects of natural and synthetic steroids and of non-steroidal antiandrogens (IC₅₀) upon the 5α-R activity were studied. Of the natural steroids, 17-0H-progesterone was found to have the highest inhibitory effect (IC₅₀=1.35 μM), followed in decreasing order by progesterone (IC₅₀=5.0 μM) and 4-androstene-3,17-dione (IC₅₀=21.6 μM). Oestradiol-17β had practically no inhibitory effect. Of the synthetic steroids, 4-MA had the highest inhibitory effect (IC₅₀=0.068 μM), followed by nortestosterone (IC₅₀=7.4 μM) and RU-486 (Mifepristone) (IC₅₀=115 μM). Even at 1000 μM, cyproterone acetate exerted no inhibitory effect. Of the nonsteroidal compounds, ketoconazole proved a weak inhibitor (IC₅₀=115 μM), while flutamide was practically ineffective. Preliminary results were presented at the 12th Congress of the Hungarian Society of Endocrinology and Metabolism, Budapest, April 1988.     

Ketamine and its isomers have equipotent relaxant effects on tracheal smooth muscle contracted by tachykinins

Recent studies indicate that not only inflammatory cells but also neural mechanisms by which tachykinins such as substance P (SP) and neurokinin A (NKA) are released from vagal afferent C-fiber contribute to asthma. Although ketamine (K) has been used in the anesthetic management of asthmatic patients, the mechanism by which K relaxes the airway smooth muscle is still uncertain, and no information exists on any differential effect of K and its isomers. We determined the spasmolytic effect of racemic [R(±)]K and its isomers S(+) K and R(−) K on SP and NKA-induced contraction of tracheal smooth muscle in guinea pigs. Strips of guinea pig trachea were mounted in an organ bath filled with Tyrode's solution at 37°C bubbled with 95% O₂/5% CO₂. Strip tension was measured isometrically with a force displacement transducer. Strip contraction was elicited with SP 10⁻⁶ M or NKA 5×10⁻⁷ M.R(±), R(−), or S(+) K (4.5−18.0×10⁻⁴M) was cumulatively administered into the bath. The calculated ED₅₀ values (the concentration that relaxed the contraction by 50%) of R(±), R(−) and S(+) K were 7.6±0.5, 7.8±0.6, and 7.6±0.5 (10⁻⁴M), respectively, when the contraction was elicited with SP, and 8.0±1.0, 8.2±1.2, and 7.9±1.3 (10⁻⁴M), respectively, when NKA was used. We concluded that K and its isomers have equipotent spasmolytic effects on airway smooth muscle precontracted with tachykinins.     

Feasibility of Intraoperative Neuromonitoring During Thyroid Surgery After Administration of Nondepolarizing Neuromuscular Blocking Agents

Background  A short-acting depolarizing neuromuscular blocking agent (NMBA), succinylcholine, has been utilized for thyroid operations with intraoperative neuromonitoring (IONM). Because of its potential to cause serious side effects, this prospective study tried to determine the feasibility of IONM after administration of a nondepolarizing NMBA during thyroid operations. Methods  Complete IONM data for 179 patients who had normal cord mobility were investigated: 90 patients received an induction dose of rocuronium (group R) and 89 received atracurium (group A). Electromyography signals were obtained from the vagus nerve before and after resection of the thyroid lobe and were defined as V₁ and V₂ signals, respectively. Accelerometry (percent twitch) was used to monitor the quantitative degree of neuromuscular blockade. Results  V₁ and V₂ signals were obtained successfully in all patients. The percent twitch at the V₁ signal was significantly lower than that at the V₂ signal in both groups (39% ± 20% vs. 69% ± 26% in group R; 35% ± 28% vs. 56% ± 35 % in group A; both p < 0.01). However, the magnitude of the V₁ and V₂ signals did not differ significantly in either in group (473.8 ± 290.8 μV vs. 528 ± 316.2 μV in group R; 584.8 ± 394.3 μV vs. 637.8 ± 458.2 μV in group A; both p > 0.05). Conclusions  A single dose of either rocuronium or atracurium was feasible for IONM during thyroid surgery and provided adequate muscle relaxation for tracheal intubation.     

Landiolol has a less potent negative inotropic effect than esmolol in isolated rabbit hearts

Purpose  We compared the negative chronotropic and inotropic effects of landiolol and esmolol, two clinically available short-acting β1-blockers with high β1-selectivity, using whole isolated rabbit heart preparations. Methods  Tachycardia was induced by continuous perfusion of 10⁻⁷ M isoproterenol, and we used concentrations of landiolol or esmolol in ascending steps (1 · 10⁻⁶, 3 · 10⁻⁶, 1 · 10⁻⁵, 3 · 10⁻⁵, and 1 · 10⁻⁴ M). Heart rate (HR), left ventricular developed pressure (LVDP), the maximal rates of left ventricular force development (LVdP/dt_max), and myocardial oxygen consumption (MVO₂) were measured and compared. Results  Both landiolol and esmolol produced dosedependent decreases in HR, LVDP, LVdP/dt_max, and MVO₂. The HR lowering effects of the two agents were comparable. At concentrations of 3 · 10⁻⁵ and 1 · 10⁻⁴ M, esmolol produced more profound depression of LVDP (47 ± 26 and 12 ± 11 mmHg, respectively; mean ± SD) and reduction of LVdP/dt_max (650 ± 287 and 120 ± 103 mmHg·s⁻¹) than landiolol (68 ± 20 and 64 ± 20 mmHg, and 897 ± 236 and 852 ± 240 mmHg·s⁻¹, respectively). At the same concentrations, esmolol caused more profound reduction in MVO₂ (40 ± 11 and 35 ± 10 μl·min⁻¹ · g⁻¹) than landiolol (50 ± 8 and 48 ± 8 μl·min⁻¹ · g⁻¹), respectively. Conclusion  Our results indicate that in the isolated rabbit heart, landiolol and esmolol had equipotent negative chronotropic effects, however, landiolol had a less potent negative inotropic effect than esmolol.     

A randomized study of the effects of perioperative i.v. lidocaine on hemodynamic and hormonal responses for cesarean section

Purpose  Intravenous infusion of lidocaine attenuates the stress response to surgery. We aimed to evaluate the effects of perioperative lidocaine on the hemodynamic and hormonal responses for cesarean delivery. Methods  After the gaining of ethical approval, 90 patients scheduled for elective cesarean delivery were randomly allocated to receive either lidocaine 1.5 mg·kg⁻¹ i.v. bolus 30 min before induction, followed by an infusion of 1.5 mg·kg⁻¹·h⁻¹ until 1 h after surgery (n = 45), or saline placebo (n = 45). Anesthesia was maintained with 50% nitrous oxide in oxygen with 0.7% isoflurane. Hemodynamic variables, plasma cortisol, maternal and neonatal lidocaine concentrations, Apgar scores at 1 and 5 min, neonatal acid-base status, and the neurologic and adaptive capacity score (NACS) were recorded. Results  After induction, patients receiving lidocaine had a smaller increase in heart rate and mean arterial blood pressure (P < 0.02) and lower plasma cortisol concentrations (31.1 ± 9.91 vs 45.6 ± 8.43 μg·dL⁻¹; P < 0.001). There were no differences between the two groups in Apgar scores, NACS, or neonatal acid-base status. After delivery, maternal and umbilical venous concentrations and umbilical vein-to-maternal vein ratios of lidocaine were 2.05 ± 0.42 μg·mL⁻ and 1.06 ± 0.31 μg·mL⁻¹, and 0.52 ± 0.07, respectively. Conclusion  Perioperative lidocaine is safe and effective in attenuating the maternal stress response to surgery for cesarean delivery.     

Direct measurement of acetylcholine release in detrusor smooth muscles isolated from rabbits

In the present study, we measured acetylcholine (ACh) released from rabbit detrusor smooth muscle strips induced by electrical field stimulation (EFS) using high-performance liquid chromatography coupled with microdialysis procedure. There were frequency- and duration-dependent increases in contractile response and ACh release. There was a significant, but not simple correlation between EFS-induced contraction and ACh release. Atropine caused a decrease and increase in the contractile response and ACh release, respectively. Pretreatment with propranolol increased ACh release, but pretreatment with phentolamine had no significant effect. These results demonstrate that this method is applicable to direct measurement of ACh release by EFS, and that neurotransmitters other than ACh may relate to EFS-induced contraction. In addition, it is suggested that there are prejunctional inhibitory muscarinic receptors and beta-adrenoceptors, which contribute to ACh release induced by EFS in the rabbit detrusor smooth muscles. Received: 20 January 1998 / Accepted: 17 April 1998     

Urinary beta2-microglobulin as a marker for vesicoureteral reflux

To determine whether urinary beta₂-microglobulin (β₂ M) excretion would be elevated in patients with severity of vesicoureteral reflux, urinary β₂ M/creatinine (Cr) ratios were measured on random urine samples in 56 children with various grades of reflux. Results were compared with ratios of 39 nonrefluxing patients matched for age and gender. Patients with evidence of renal insufficiency or urinary tract infection were excluded. Bladder urine was obtained at the time of the vesicoureterogram. Reflux was graded using the International Reflux Classification System. The mean urinary β₂ M/Cr ratio was higher in the refluxing group (1.82±0.6 μg/mg Cr) than in the nonrefluxing control group (0.54±0.09 μg/mg Cr, P <0.01). When the mean urinary β₂ M/Cr ratios were compared for each grade of reflux with the nonrefluxing control group, patients with grade IV and V reflux had significantly higher urinary β₂ M/Cr values than the nonrefluxing patients (2.83±0.71 μg/mg Cr and 4.61±0.65 μg/mg Cr, P <0.001, respectively). No patient with grade I, II, or III reflux had a urinary β₂ M/Cr ratio above 0.92 μg/mg Cr. Statistical analysis revealed no significant differences among the mean β₂ M/Cr ratio for grade I (0.53±0.08), II (0.51±0.09), or III (0.59±0.17) refluxers or the nonrefluxing controls. Therefore, urinary β₂ M/Cr ratios are increased in children with a high grade of reflux. Such values may be useful in the early detection of tubular damage in patients with vesicoureteral reflux. Received June 23, 1995; received in revised form and accepted January 17, 1996     

Succinylcholine potentiates acetylcholine-induced contractile and phosphatidylinositol responses of rat trachea

Purpose Although succinylcholine (SCh) is often used as a muscle relaxant in electroconvulsive therapy, its influence on airway reactivity has not been fully investigated. We examined the effects of SCh on acetylcholine (ACh)-, carbachol (CCh)-, and electrical field stimulation (EFS)-induced contractions, and on the ACh-induced phosphatidylinositol (PI) response of rat trachea. Methods Thirty-two male Wistar rats weighing 250–350 g were used. The trachea was rapidly isolated and cut into 3-mm-wide rings. The resting tension was adjusted periodically to 1.0 g during the equilibration period. ACh, 1 μM; carbachol (CCh), 0.05 μM; or neither of them, was added, and SCh was then added at 1–300 μM final concentrations, and ring tension was examined. Contractions were elicited by EFS in the presence or absence of 100 μM SCh. Tracheal slices were incubated with [³H] myo-inositol, 1 μM ACh, and various concentrations of SCh. The accumulation of [³H] inositol monophosphate (IP₁) was measured. Results SCh did not affect the tension by itself without ACh, or with CCh, but SCh potentiated the ACh-induced contraction of rat trachea at concentrations of 10 μM or more (50% effective concentration [EC₅₀]; 43.6 μM). SCh produced a significant increase in the amplitude and duration of EFS-induced contractions. SCh, at concentrations of 10 μM and 100 μM, potentiated ACh-induced IP₁ accumulation. Conclusion SCh potentiated ACh-induced, but not CCh-induced, contractile and PI responses, and enhanced EFS-induced contraction of rat trachea, suggesting that competition for butyrylcholinesterase (BChE) in airway smooth muscle could be involved in the potentiation by SCh of ACh-induced airway smooth muscle contraction.     

Skeletal adenylate cyclase: Effect of Mg2+, Ca2+, and PTH

Summary Plasma membranes were prepared from mineralized guinea pig bone in order to study Mg²⁺ and Ca²⁺ modulation of skeletal adenylate cyclase. Plasma membrane preparation was accomplished by crushing the bone in liquid N₂ and subsequent multiple washings in buffer containing EGTA to remove all Ca²⁺ prior to adenylate cyclase assay. Skeletal adenylate cyclase was found to be dependent on GTP and Mg²⁺ and responsive to bovine 1-34 PTH. Ca²⁺ caused a competitive inhibition of Mg²⁺-activated skeletal adenylate cyclase. The apparent KaMg was 1.9±0.3 in the presence of 0.2 μM Ca²⁺ but increased to a mean of 7.2±1.3 in the presence of 5.0 μM Ca²⁺. Analysis of the Ca²⁺ inhibition curves at concentrations from .05 μM-1.0 mM were consistent with the presence of two Ca²⁺ inhibition sites, one with an apparent Ki of 1–2 μM and the other with an apparent Ki of approximately 500 μM. Lowering the Mg²⁺ concentration increased the contribution of the high affinity Ca²⁺ binding site to the overall Ca²⁺ inhibition, and raising the Mg²⁺ concentration had the opposite effect. While bPTH 1-34 enhanced adenylate cyclase activity, it did not increase the affinity of Mg²⁺ for skeletal adenylate cyclase nor did it alter the K_iCa or the pattern of Ca²⁺ inhibition. These data may explain the skeletal resistance to PTH during Mg deficiency. A fall in the intracellular Mg would render the adenylate cyclase more susceptible to inhibition by the prevailing intracellular Ca²⁺ concentration. Since PTH does not appear to modulate either Mg²⁺ activation or Ca²⁺ inhibition of skeletal adenylate cyclase, cAMP-mediated PTH induction of bone resorption would be impaired, and hypocalcemia would occur.     

Intestinal Afferent Nerve Sensitivity is Increased During the Initial Development of Postoperative Ileus in Mice

Introduction  Neuronal reflex inhibition of gastrointestinal motility is a key mechanism in the development of postoperative ileus (POI). The aim of our study was to determine whether intestinal afferent nerve fibers are sensitized during the first hours after surgery contributing to this mechanism. Methods  Under enflurane anesthesia, C57BL/6 mice underwent laparotomy followed by sham treatment or standardized small bowel manipulation to induce POI. After 1, 3, or 9 h, extracellular multi-unit mesenteric afferent nerve recordings were performed in vitro from 2 cm segments of jejunum (subgroups n = 6) superfused with Kreb’s buffer (32°C, gassed with O₂/CO₂ mixture). Segments were cannulated to monitor luminal pressure and intestinal motility. Afferent impulses as response to bradykinin (0.5 μM) and to mechanical ramp distension of the intestinal lumen from 0 to 80 cmH₂O were recorded. Results  At 1 h, amplitudes of intestinal contractions were 0.8 ± 0.2 cmH₂O after induction of POI and 5.0 ± 0.8 cmH₂O in sham controls (mean ± SEM; p < 0.01). A similar difference was observed for segments harvested at 3 and 9 h. Afferent firing to serosal bradykinin was increased at 1, 3, and 9 h in POI segments compared to sham controls (p < 0.05 at 1 h, p < 0.01 at 3 and 9 h). During distension with high pressures, afferent firing rate was increased at 1 and 3 h in segments after induction of POI compared to sham controls. Nine hours postoperatively, contracted and dilated segments were observed during POI that were investigated separately. While afferent firing in dilated segments was increased to 176 ± 16 imp s⁻¹ at 80 cmH₂O luminal distension (p < 0.01), it was 46 ± 5 imp s⁻¹ in contracted segments (p < 0.001) compared to 77 ± 4 imp s⁻¹ in sham controls. Conclusions  Afferent firing to bradykinin and high threshold distension is augmented in the early phase of POI. As these stimuli are known to sensitize predominantly spinal afferents, this mechanism may contribute to reflex inhibition of intestinal motility during POI.     

Evaluation of glucose tolerance,insulin secretion,and insulin action in patients with primary hyperparathyroidism before and after surgery

Summary Glucose tolerance, insulin secretion, and insulin sensitivity were evaluated in 8 asymptomatic patients with primary hyperparathyroidism (PHPT) before and at least 8 weeks after surgical correction of PHPT by means of the hyperglycemic clamp technique. In addition, 15 sex- and agematched control subjects were investigated for comparative reasons by the same technique. Glucose metabolized (M) during the hyperglycemic clamp was not significantly (NS) different between patients with PHPT and controls (7.9±2.3 vs. 6.3±1.9 mg/kg/min). However, insulin secretion (I) was significantly elevated in patients with PHPT compared to controls (87±17 vs. 45±12 μU/ml,P<0.05). The calculated insulin sensitivity index, (M/I) was significantly reduced in PHPT compared to controls (11.0±2.1 vs. 15.2±1.4 mg/kg/min per μU/ml×100,P<0.05). Comparing patients with PHPT before and after surgery, the M value, which is a measure of glucose tolerance, was not significantly different (7.9±2.3 vs. 7.8±1.5 mg/kg/min). However, insulin secretion was significantly lower after surgical correction of PHPT compared to the preoperative situation (48±9 μU/ml vs. 87±17 μU/7 ml,P<0.01). The calculated M/I rose significantly after surgery compared to the preoperative value (11±2.1 vs. 17.6±2.7 mg/kg/min per μU/ml ×100,P<0.001). We conclude that disturbed carbohydrate metabolism such as insulin hypersecretion and insulin resistance, in patients with PHPT is an early finding in this disease and that these early disturbances in glucose metabolism are, however, fully reversible. Correction of disturbed carbohydrate metabolism in PHPT might be a distinct argument for early surgical intervention in this disease.     

Role of Selective α and β Adrenergic Receptor Mechanisms in Rat Jejunal Longitudinal Muscle Contractility

Gut motility is modulated by adrenergic mechanisms. The aim of our study was to examine mechanisms of selective adrenergic receptors in rat jejunum. Spontaneous contractile activity of longitudinal muscle strips from rat jejunum was measured in 5-ml tissue chambers. Dose–responses (six doses, 10⁻⁷–3 × 10⁻⁵M) to norepinephrine (NE, nonspecific), phenylephrine (PH, α₁), clonidine (C, α₂), prenalterol (PR, β₁), ritodrine (RI, β₂), and ZD7714 (ZD, β₃) were evaluated with and without tetrodotoxin (TTX, nerve blocker). NE(3 × 10⁻⁵M) inhibited 74 ± 5% (mean ± SEM) of spontaneous activity. This was the maximum effect. The same dose of RI(β₂), PH(α₁), or ZD(β₃) resulted in an inhibition of only 56 ± 5, 43 ± 4, 33 ± 6, respectively. The calculated concentration to induce 50% inhibition (EC50) of ZD(β₃) was similar to NE, whereas higher concentrations of PH(α₁) or RI(β₂) were required. C(α₂) and PR(β₁) had no effect. TTX changed exclusively the EC50 of RI from 4.4 ± 0.2 to 2.7 ± 0.8% (p < 0.04). Contractility was inhibited by NE (nonspecific). PH(α₁), RI(β₂), and ZD(β₃) mimic the effect of NE. TTX reduced the inhibition by RI. Our results suggest that muscular α₁, β₂, and β₃ receptor mechanisms mediate adrenergic inhibition of contractility in rat jejunum. β₂ mechanisms seem to involve also neural pathways. Part of this work was presented as a poster at the annual meeting of the Society for Surgery of the Alimentary Tract, Orlando, FL, May 17–22, 2003, and published as an abstract in Gastroenterology 2003, 124(4):M1342.     

Functional analysis of ryanodine receptor type 1 p.R2508C mutation in exon 47

Purpose  Malignant hyperthermia (MH) is a pharmacogenetic disorder of intracellular calcium homeostasis with an autosomal dominant inheritance. Most of the reported mutations in exon 47 were identified in Asian patients. However, no functional analysis of p.R2508C has been performed. We therefore conducted a functional analysis of the mutation by altering calcium homeostasis in human embryonic kidney (HEK) 293 cells transfected with the p.R2508C mutation in exon 47 of the ryanodine receptor 1 (RYR1). Methods  The entire RYR1 coding region from genomic DNA, which was extracted from the biopsied muscle specimens of two patients, was sequenced. The p.R2508C mutation was introduced into rabbit RYR1 cDNA, and wild-type or p.R2508C mutant cDNAs were transfected into HEK-293 cells. Using the calcium-sensitive probe Fura 2, we utilized the 340/380 nm ratio to analyze alterations in calcium homeostasis following treatment with caffeine and 4-chloro-m-cresol (4CmC). Results  Genetic analysis revealed a C→T point mutation of RYR1 exon 47 at position 7522, resulting in an amino acid exchange of arginine for cysteine at amino acid 2508. The half-maximal activation concentrations (EC₅₀) of caffeine and 4CmC for HEK-293 cells transfected with the p.R2508C mutation were 1.86 ± 0.23 mM and 73.14 ± 19.44 μM, while those for wild-type RYR1 were 2.62 ± 0.23 mM and 179.31 ± 35.23 μM, respectively. Conclusion  We demonstrated that the transfected RYR1 mutant was more sensitive to caffeine and 4CmC than wildtype RYR1. These findings suggest that the p.R2508C mutation may be pathogenetic for susceptibility to MH.     

Human pharmacokinetics of orally administered strontium

Summary Pharmacokinetics of orally administered SrCl₂ (2.5 mmol), were studied in six healthy male volunteers. In the overall plasma concentration time (C-t) curves, two absorption phases were observed due to two dominant intestinal absorption loci. A method was devised to obtain separately the plasma C-t curves associated with each of the two absorption loci (curve 1 and curve 2). These curves and the overall plasma C-t curve were analyzed with a nonlinear estimation program (PCNONLIN). Pharmacokinetic parameters (mean±SD, n=6) calculated from the overall curve were as follows: peak plasma concentration (C_max) 3.55±1.22 μg/ml and area under the plasma C-t curve (AUC^∞) 9138±1930 μg·min/ml. The pharmacokinetic parameters calculated from curve 1 were as follows: terminal plasma elimination half-life time 47.3±7.9 hour, the plasma elimination half-life time of the preceding phase 5.2±3.3 hour, C_max,1 3.09±0.95 μg/ml, the first-order absorption rate constant for absorption locus 1 (k_a,1) 5.7±1.2×10⁻² minute⁻¹ and the time lag (t_{lag, 1}) 11.7±7.9 minute. In three of the subjects the pharmacokinetic parameters of absorption locus 2 could be evaluated: k_a,2=4.6±0.4×10⁻² minute⁻¹, t_lag,2=77.3±4.0 minute, t_max,2=153±16 minute, C_max,2=0.9±0.4μg/ml and AUC ₂ ^∞ =1204±565 μg·minute/ml. AUC ₂ ^∞ /AUC^∞=0.14, indicating that 14% of the absorbed dose was absorbed via the second locus. The half-life time for the urinary strontium (Sr):creatinine ratio was 39.5±9.5 hour and the cumulative urinary excretion (day 0–6) was 34.0±13.8 mg, representing 15.5±6.3% of the administered dose and 84% of the estimated absorbed dose, respectively. The estimated bioavailability was 20% and the renal clearance (day 0–6) was lower than 4 ml/minute, indicating tubular reabsorption of Sr.