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Serotonin 5-HT(2C) receptors represent targets for therapeutics aimed at treating anxiety, depression, schizophrenia, and obesity. Previously, we demonstrated that 5-HT(2C) receptors function as homodimers. Herein, we investigated the effect of agonist and inverse agonist treatment on the homodimer status of two naturally occurring 5-HT(2C) receptor isoforms, one without basal activity (VGV) and one with constitutive activity (INI) with respect to Galpha(q) signaling. Cyan- and yellow-fluorescent proteins were used to monitor VGV and INI homodimer formation by western blot, and in living cells using bioluminescence and fluorescence resonance energy transfer (BRET and FRET). Western blots of solubilized membrane proteins revealed equal proportions of homodimeric receptor species from HEK293 cells transfected with either the VGV or INI isoform in the absence and presence of 5-HT. BRET ratios measured in HEK293 cells transfected with the VGV or INI isoform were the same and were not modulated by 5-HT. Similarly, FRET efficiencies were the same regardless of whether measured in cells expressing the VGV or INI isoform in the absence or presence of 5-HT or clozapine. The results indicate that serotonin 5-HT(2C) receptors form homodimers regardless of whether they are in an inactive or active conformation and are not regulated by drug treatment.

Dimerization is a common property of G-protein-coupled receptors (GPCR). While the formation of GPCR dimers/oligomers has been reported to play important roles in regulating receptor expression, ligand binding, and second messenger activation, less is known about how and where GPCR dimerization occurs. The present study was performed to identify the precise cellular compartment in which class A GPCR dimer/oligomer biogenesis occurs. We addressed this issue using confocal microscopy and fluorescence resonance energy transfer (FRET) to monitor GPCR proximity within discrete intracellular compartments of intact living cells. Time-lapse confocal imaging was used to follow CFP- and YFP-tagged serotonin 5-HT2C receptors during biosynthesis in the endoplasmic reticulum (ER), trafficking through the Golgi apparatus and subsequent expression on the plasma membrane. Real-time monitoring of FRET between CFP- and YFP-tagged 5-HT2C receptors was performed by acceptor photobleaching within discrete regions of the ER, Golgi, and plasma membrane. The FRET signal was dependent on the ratio of CFP- to YFP-tagged 5-HT2C receptors expressed in each region and was independent of receptor expression level, as predicted for proteins in a non-random, clustered distribution. FRET efficiencies measured in the ER, Golgi, and plasma membrane were similar. These experiments provide direct evidence for homodimerization/oligomerization of class A GPCR in the ER and Golgi of intact living cells, and suggest that dimer/oligomer formation is a naturally occurring step in 5-HT2C receptor maturation and processing.

This chapter describes successful assignments that made creative use of laptops in writing, literature, and public speaking courses. Some activities moved the session out of the classroom to outdoor locations.

This chapter describes Clemson University's Laptop Faculty Development Program and its assessment, offering the program as one model for designing faculty development to successfully implement laptop mandates. The chapter also acquaints readers with the many types of in-class, laptop-based activities that meet best-practice criteria for effective teaching.

Background— The formation of angiotensin-(1-7) from either angiotensin (Ang) I or Ang II in failing human hearts is not well understood. Methods and Results— Angiotensinase activity in left and right ventricular membranes from 14 idiopathic dilated cardiomyopathy (IDC), 8 primary pulmonary hypertension (PPH), and 13 nonfailing human hearts was measured with either 125 I-Ang I or 125 I-Ang II as substrate. Ang-(1-7)–forming activity from 125 I-Ang I was inhibited by thiorphan. With 125 I-Ang II as substrate, Ang-(1-7) formation was inhibited by the ACE2-specific inhibitor C16. Western blotting with an anti-ACE2 antibody confirmed the presence of ACE2. Angiotensinase activity with 125 I-Ang I as substrate was increased in failing IDC left ventricles (LVs) compared with nonfailing LVs ( P 125 I-Ang II as substrate was increased in both failing LVs and right ventricles (RVs) of IDC hearts and only in failing RVs of PPH hearts (PPH LV, 51.12±5.25; PPH RV, 89.97±11.21; IDC LV, 139.7±21.96; and IDC RV, 192.7±5.43; NF LV, 32.89±5.38; NF RV 40.49±10.66 fmol/min per milligram ( P P P P Conclusions— Ang-(1-7)–forming activity from both Ang I and Ang II was increased in failing human heart ventricles but was mediated by at least two different angiotensinases. The first, which demonstrated substrate preference for Ang I, was neutral endopeptidase (NEP)-like. The second was ACE2, as demonstrated by Western blotting and inhibition of activity with C16.

We previously described a negative allosteric modulator (NAM) of FSHR (ADX61623) that blocked FSH-induced cAMP and progesterone production but did not block estradiol production. That FSHR NAM did not affect FSH-induced preovulatory follicle development as evidenced by the lack of an effect on the number of FSH-dependent oocytes found in the ampullae following ovulation with hCG. A goal is the development of a nonsteroidal contraceptive. Toward this end, a high-throughput screen using human FSHR identified an additional nonsteroidal small molecule (ADX68692). Although ADX68692 behaved like ADX61623 in inhibiting production of cAMP and progesterone, it also inhibited FSH-induced estradiol in an in vitro rat granulosa primary cell culture bioassay. When immature, noncycling female rats were injected subcutaneously or by oral dosing prior to exogenous FSH administration, it was found that ADX68692 decreased the number of oocytes recovered from the ampullae. The estrous cycles of mature female rats were disrupted by administration by oral gavage of 25 mg/kg and 10 mg/kg ADX68692. In the highest dose tested (25 mg/kg), 55% of animals cohabited with mature males had implantation sites compared to 33% in the 10 mg/kg group and 77% in the control group. A surprising finding was that a structural analog ADX68693, while effectively blocking progesterone production with similar efficacy as ADX68692, did not block estrogen production and despite better oral availability did not decrease the number of oocytes found in the ampullae even when used at 100 mg/kg. These data demonstrate that because of biased antagonism of the FSHR, nonsteroidal contraception requires that both arms of the FSHR steroidogenic pathway must be effectively blocked, particularly estrogen biosynthesis. Thus, a corollary to these findings is that it seems reasonable to propose that the estrogen-dependent diseases such as endometriosis may benefit from inhibition of FSH action at the ovary using the FSHR NAM approach.

We previously described a negative allosteric modulator (NAM) of FSHR (ADX61623) that blocked FSH-induced cAMP and progesterone production but did not block estradiol production. That FSHR NAM did not affect FSH-induced preovulatory follicle development as evidenced by the lack of an effect on the number of FSH-dependent oocytes found in the ampullae following ovulation with hCG. A goal is the development of a nonsteroidal contraceptive. Toward this end, a high-throughput screen using human FSHR identified an additional nonsteroidal small molecule (ADX68692). Although ADX68692 behaved like ADX61623 in inhibiting production of cAMP and progesterone, it also inhibited FSH-induced estradiol in an in vitro rat granulosa primary cell culture bioassay. When immature, noncycling female rats were injected subcutaneously or by oral dosing prior to exogenous FSH administration, it was found that ADX68692 decreased the number of oocytes recovered from the ampullae. The estrous cycles of mature female rats were disrupted by administration by oral gavage of 25 mg/kg and 10 mg/kg ADX68692. In the highest dose tested (25 mg/kg), 55% of animals cohabited with mature males had implantation sites compared to 33% in the 10 mg/kg group and 77% in the control group. A surprising finding was that a structural analog ADX68693, while effectively blocking progesterone production with similar efficacy as ADX68692, did not block estrogen production and despite better oral availability did not decrease the number of oocytes found in the ampullae even when used at 100 mg/kg. These data demonstrate that because of biased antagonism of the FSHR, nonsteroidal contraception requires that both arms of the FSHR steroidogenic pathway must be effectively blocked, particularly estrogen biosynthesis. Thus, a corollary to these findings is that it seems reasonable to propose that the estrogen-dependent diseases such as endometriosis may benefit from inhibition of FSH action at the ovary using the FSHR NAM approach.

State libraries in Rhode Island and New Jersey have found ways to cut costs by outsourcing delivery services and, in Rhode Island, the operation of the Regional Library for the Blind and Physically Handicapped.

High quality gamete production in males and females requires the pituitary gonadotropin follicle stimulating hormone (FSH). In this report a novel chemical class of small molecule inhibitors of FSH receptor (FSHR) is described. ADX61623, a negative allosteric modulator (NAM), increased the affinity of interaction between (125)I-hFSH and human FSHR (hFSHR) five fold. This form of FSHR occupied simultaneously by FSH and ADX61623 was inactive for cAMP and progesterone production in primary cultures of rat granulosa cells. In contrast, ADX61623 did not block estrogen production. This demonstrates for the first time, biased antagonism at the FSHR. To determine if ADX61623 blocked FSH induction of follicle development in vivo, a bioassay to measure follicular development and oocyte production in immature female rats was validated. ADX61623 was not completely effective in blocking FSH induced follicular development in vivo at doses up to 100mg/kg as oocyte production and ovarian weight gain were only moderately reduced. These data illustrate that FSHR couples to multiple signaling pathways in vivo. Suppression of one pool of FSHR uncouples Gαs and cAMP production, and decreases progesterone production. Occupancy of another pool of FSHR sensitizes granulosa cells to FSH induced estradiol production. Therefore, ADX61623 is a useful tool to investigate further the mechanism of the FSHR signaling dichotomy. This may lead to a greater understanding of the signaling infrastructure which enables estrogen biosynthesis and may prove useful in treating estrogen dependent disease.

Histamine stimulation of swine arterial smooth muscle is associated with a high [Ca2+]i sensitivity for increases in myosin light-chain phosphorylation. In contrast, KCl depolarization produces a relatively lower [Ca2+]i sensitivity (i.e., similar increases in [Ca2+]i induce less myosin phosphorylation). We evaluated whether 1) artifacts in the methodology for measuring [Ca2+]i or 2) true alterations in the [Ca2+]i sensitivity of myosin light-chain kinase were responsible for these apparent changes in the [Ca2+]i sensitivity of phosphorylation. The [Ca2+]i sensitivity of phosphorylation was higher with histamine stimulation regardless of whether the [Ca2+]i indicator was aequorin (which was loaded intracellularly by reversible hyperpermeabilization) or Fura 2 (which was loaded intracellularly by incubation of the tissues in Fura 2 AM). Aequorin and Fura 2 appeared to detect qualitatively similar stimulus-induced changes in [Ca2+]i with the exception that the initial response to histamine stimulation was different (histamine initially induced a large aequorin light transient and a relatively smaller increase in Fura 2 fluorescence). The [Ca2+]i sensitivity of myosin light-chain kinase extracted from KCl depolarized tissues was lower than the [Ca2+]i sensitivity of myosin light-chain kinase extracted from unstimulated or histamine stimulated tissues. These results suggest that depolarization specifically modifies myosin light-chain kinase to decrease its [Ca2+]i sensitivity. Changes in the [Ca2+]i sensitivity of myosin light-chain phosphorylation are not an artifact of the [Ca2+]i measurement technique.

The contractile sensitivity of smooth muscle to changes in myoplasmic [Ca2+] is dependent on the form of stimulation. Both myosin phosphorylation and force are less sensitive to increases in [Ca2+]i derived from Ca2+ entry through L-type Ca2+ channels than to increases in [Ca2+] induced by agents which release internal Ca2+ stores. We hypothesized that activation of receptor-operated channels should produce a [Ca2+]i sensitivity similar to that induced by opening L channels. Aequorin-estimated myoplasmic [Ca2+] and myosin light chain phosphorylation were measured in swine carotid media tissues stimulated with ATP, an activator of the only known receptor-operated cation channel in smooth muscle. ATP, via activation of a P2x purinergic receptor, induced large, transient increases in [Ca2+]i, yet only small transient elevations in phosphorylation or force. Rapid desensitization to ATP was partially, but not completely, caused by hydrolysis of ATP into adenosine since 1) alpha-beta-methylene ATP (a poorly hydrolyzable analog of ATP) produced larger, yet still transient increases in [Ca2+]i, phosphorylation, and force; 2) BW A1433U, a P1 (adenosine) receptor antagonist, enhanced ATP-induced contractions; and 3) ATP, but not alpha-beta-methylene ATP increased bath [adenosine]. The [Ca2+]i sensitivity of phosphorylation during P2x receptor activation was similar to that observed with KCl-depolarization-induced opening of L channels, supporting the hypothesis that transplasmalemmal Ca2+ influx produces less phosphorylation and force than mobilization of intracellular Ca2+ stores. Cumulative additions of higher alpha-beta-methylene ATP concentrations induced repeated transient contractions, indicative of an unusual form of receptor desensitization which could be explained if the affinity of the P2x receptor for ATP, but not the receptor number were rapidly reduced.

Dimerization is a common property of G-protein-coupled receptors (GPCR). While the formation of GPCR dimers/oligomers has been reported to play important roles in regulating receptor expression, ligand binding, and second messenger activation, less is known about how and where GPCR dimerization occurs. The present study was performed to identify the precise cellular compartment in which class A GPCR dimer/oligomer biogenesis occurs. We addressed this issue using confocal microscopy and fluorescence resonance energy transfer (FRET) to monitor GPCR proximity within discrete intracellular compartments of intact living cells. Time-lapse confocal imaging was used to follow CFP- and YFP-tagged serotonin 5-HT2C receptors during biosynthesis in the endoplasmic reticulum (ER), trafficking through the Golgi apparatus and subsequent expression on the plasma membrane. Real-time monitoring of FRET between CFP- and YFP-tagged 5-HT2C receptors was performed by acceptor photobleaching within discrete regions of the ER, Golgi, and plasma membrane. The FRET signal was dependent on the ratio of CFP- to YFP-tagged 5-HT2C receptors expressed in each region and was independent of receptor expression level, as predicted for proteins in a non-random, clustered distribution. FRET efficiencies measured in the ER, Golgi, and plasma membrane were similar. These experiments provide direct evidence for homodimerization/oligomerization of class A GPCR in the ER and Golgi of intact living cells, and suggest that dimer/oligomer formation is a naturally occurring step in 5-HT2C receptor maturation and processing.

Regulation of [Ca2+]i and modulation of the [Ca2+]i sensitivity of myosin phosphorylation in smooth muscles may involve phosphorylation of one or more proteins on tyrosine residues. We tested this hypothesis by measuring tyrosine phosphorylation of proteins extracted from swine carotid artery and separated by SDS gel electrophoresis. Tyrosine phosphorylation was estimated by the binding of antiphosphotyrosine antibodies to proteins in SDS gels. We found four bands with approximate molecular weights of 120, 110, 85 and 75 kD in which tyrosine phosphorylation increased 1 min after histamine stimulation. After washout of histamine, dephosphorylation of the proteins in these four bands occurred at a slower rate than relaxation. Tyrosine phosphorylation of these four protein bands did not correlate with the [Ca2+]i sensitivity of myosin phosphorylation following agonist or high [K+]o stimulation. Phorbol dibutyrate stimulation also induced tyrosine phosphorylation of these four protein bands. These data suggest that tyrosine phosphorylation of the proteins in these four bands may be involved in the initial phase of swine carotid artery contraction. However, there was, at most, only a minor involvement of tyrosine phosphorylation of these four protein bands in the sustained phase of contraction or in the regulation of [Ca2+]i sensitivity of myosin phosphorylation in the swine carotid artery. These data do not rule out a role for other, less abundant tyrosine phosphoproteins in the regulation of sustained contraction or the [Ca2+]i sensitivity of myosin phosphorylation.

Sustained smooth muscle contraction has been proposed to be regulated by either 1) sustained increases in intracellular Ca2+ concentration [(Ca2+]i)-dependent myosin phosphorylation or 2) diacylglycerol-dependent protein kinase C activation. We measured diacylglycerol mass with the diacylglycerol kinase assay and myoplasmic [Ca2+] with aequorin in swine carotid medial smooth muscle. Sustained and significant increases in [Ca2+], myosin light chain phosphorylation, and isometric stress were observed with histamine or endothelin stimulation. Neither stimuli, however, induced significant increases in diacylglycerol mass. Relaxation of histamine-stimulated tissues was induced by removal of histamine or removal of extracellular CaCl2 in the continued presence of histamine. The rate of decline of both [Ca2+] and force was similar in both protocols, suggesting that removal of Ca2+ (without removing the stimulus) was equivalent to removal of the stimulus. These data suggest that [Ca2+]i is the primary regulator of sustained swine arterial smooth muscle contraction, whereas diacylglycerol has, at most, only a minor role.

The antihypertensive effects of captopril and enalapril maleate were studied over a 10-week period in 24 hypertensive patients randomized into captopril or enalapril treatment groups. Prestudy blood pressure was 171 +/- 4/109 +/- 1 mm Hg and after 4 weeks of hydrochlorothiazide 160 +/- 4/103 +/- 1 (p less than 0.05). With the addition of converting enzyme inhibitor to hydrochlorothiazide the blood pressure decreased at 3 h to 132 +/- 3/87 +/- 2 in the subjects. The diastolic blood pressure decreased acutely more with captopril (-24) than with enalapril (-17) (p less than 0.05). After 10 weeks of combined therapy the depressor response was maintained (134 +/- 3/83 +/- 1) and there was no difference between the diastolic blood pressure in the two groups treated with captopril and enalapril. Acute and chronic responses of plasma renin activity, plasma aldosterone, and converting enzyme to the angiotensin-converting enzyme inhibitor were determined. There was a significant correlation between the acute fall in diastolic blood pressure and rise in plasma renin activity in patients treated with captopril but not with enalapril. In conclusion, there is an acute depressor response with converting enzyme inhibition which is more pronounced with captopril than with enalapril and which correlates with an increase in plasma renin activity. With more prolonged treatment, the two drugs show equivalent efficacy in reducing blood pressure, inhibiting angiotensin-converting enzyme, reducing aldosterone, and stimulating plasma renin activity.

SUMMARY Fructose solutions were injected into pigs of from a few hours to 17 days of age and the rate of subsequent fall in plasma fructose concentration measured. The rate of fall, which in each experiment was approximately proportional to plasma fructose level, increased with age, being too small to observe in the first day of life.

The Pharmaceutical Information Control System (PICS), developed at Merck Sharp & Dohme Research Laboratories, provides centralized control and methodology for a series of decentralized information areas in the Division. It is compatible with and instrumental in total data processing and analysis of research information. Serving as a Core Index to all information resources of the Research Laboratories, it also processes, stores, and retrieves research project information for the staff members for planning and retrospective searches. A register of all domestic and international clinical research information on experimental and in-line products of Merck & Co., Inc., is provided by the system. An eight-digit dual-faceted classification code was developed based on a companywide program identification scheme. This code of two mutually exclusive facets enables us to identify a product with a field of research. This code has been adopted for use in administrative planning, cost accounting, time allocation, and internal reporting. The uniform use of this information code within the Research Division minimizes the vocabulary barrier between the user and information system and provides the system with a self-indexing device for internal reports. Incoming mail is copied and registered by the information center prior to transmittal to the addressee. Copies of all outgoing mail and intramural correspondence are directed to the center. An information scientist analyzes each document and selects the project code and document descriptors. This information is punched into 80-column cards. The documents are filed by code and the cards are filed alphabetically by name, term, and by date. Output forms include information, documents, printouts of document citations, and reports to drug regulatory agencies. Continuous system evaluation results in reduced I/O time, better utilization of personnel, and improved user feedback and contact.

The Medical Information Center of Schering Corporation is the central depository for all original clinical documents. The Clinical Documentation Section of the Center has developed an automated document storage and retrieval system capable of generating indexes to all documents in the system, summaries, and reports for management by computer. Documents are indexed and stored according to a project-product oriented classification scheme and the resulting citations reduced to tab cards for computer input. Citation master files are updated daily, and cumulative outputs are issued. Major system sources are completely updated and generated monthly by computer and include a patient document citation index, keyword index, file index, and patient document log book. Many special outputs are issued upon request. Other system functions include providing documents for F.D.A. submissions and the maintenance of a grant-reporting system capable of generating financial and clinical status reports by computer. The system services all company divisions engaged in clinical research and offers complete and immediate availability, accessibility, and security of documents and information.

Total compliance and lower respiratory resistance, that is, total respiratory resistance less upper airway resistance, have been measured during passive expiration in anaesthetized, intubated, completely relaxed animals, lying on their sides. Nine horses and one mule were studied. Compliance was determined for each animal from the volumes expired after inflation of the lungs to various pressures. The mean compliance for the horses was 0.246 (S.D. 0.088) litre/cm H2O or 0.573 (S.D. 0.229) (ml/cm H2O)/kg body mass. Resistance was measured in the course of passive expiration through an endotracheal tube. The decline of expiratory flow deviated markedly from an exponential and the reasons for this are discussed. Except in one horse, subsequently confirmed to be emphysematous, the pressure drop P across the lower respiratory resistance could be satisfactorily related to the expiratory flow V by an equation of the form P = k1V + k2V2. The mean value of k1 was 0.54 (S.D. 0.31) cm H2O/(litre/sec) and of k2 was 0.050 (S.D. 0.042) cm H2O/(litre/sec)2.

ACE2 purified from failing human heart was found to form a complex with integrin β1 by immunoprecipitation, Western blotting, activity assay, and ESI tandem mass spectroscopy. The ACE2/integrin complex showed a Km of 6.8 μM and a Vmax of 2.13 pmol/min/μl purified enzyme. Activity was optimal at pH 7.5 with Ang II substrate.

24 hypertensive patients were randomised into 2 groups to compare the antihypertensive effects of enalapril and captopril over a 10-week period. In the hydrochlorothiazide run-in period, blood pressure was reduced from 171 +/- 4/109 +/- 1mm Hg to 160 +/- 4/103 +/- 1mm Hg (p less than 0.05). Angiotensin-converting enzyme (ACE) inhibition decreased blood pressure to 132 +/- 3/87 +/- 2mm Hg. Captopril decreased diastolic blood pressure significantly more after 3 hours than enalapril (-24 versus -17mm Hg, p less than 0.05). After 10 weeks of therapy, this antihypertensive response was maintained at 134 +/- 3/83 +/- 1mm Hg. There was no difference between the captopril and enalapril treated groups. Acute and chronic responses of plasma renin activity, plasma aldosterone and ACE were determined. There was an acute positive correlation between the rise in plasma renin activity and the fall in blood pressures with captopril but not with enalapril. With chronic treatment there was no difference in the ability of either of the 2 drugs to reduce blood pressure, inhibit ACE, reduce aldosterone or stimulate plasma renin activity.

As a preliminary investigation of the comparative effects of the neutron beam from a 45-in. cyclotron with x rays from an 8-Mev linear accelerator, an anesthetic technique that would assure complete immobilization of the animals for several hours was sought. Choice of anesthetic technique was influenced by the nature of the experimental animal, and by the need for a nonexplosive agent (because of the proximity of high voltage electrical apparatus) which would provide safe anesthesia for long periods by remote control. For premedication, phenothiazine derivatives were chosen because they produce sedation without undue respiratory depression and because their effects are long lasting. Promazine (2 mg) and promethazine (1 mg per lb (0.45 kg) body weight) were given together by intramuscular injection 11/2 hours before induction of anesthesia. Parasympathetic depression was obtained with atropine sulfate (0.32 mg given by intramuscular injection after induction). Anesthesia was induced with intravenous thiopentone (250 mg for a 50-lb pig). On some occasions, to reduce the number of venepunctures, anesthesia was induced with cyclopropane. Pig and anesthetic apparatus were conveyed into the linear accelerator chamber, where the animal was irradiated with 8-Mev x rays. The whole process of anesthesia, transfer, positioning and irradiation took 5 tomore » 6 hr, and during much of this time the animal was inaccessible to the operating team. An optimal level of anesthesia was maintained by observing a stethograph which recorded the pig's respiratory changes. Rises in body temperature during irradiation were noted in some instances, and this was subsequently avoided by reducing ambient temperature and humidity.« less