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Tropifexor, a non-bile acid farnesoid X receptor (FXR) agonist, has dose-proportional pharmacokinetics (PK) and no obvious major enterohepatic circulation. This open-label study investigated the effect of hepatic impairment (HI), as determined by Child-Pugh grade, on tropifexor's PK, safety, and tolerability following a 200-μg dose in the fasted state. Blood samples were collected through 168 hours after dosing for quantification and plasma protein-binding determination. Total tropifexor exposure was comparable across participants with HI vs those with normal hepatic function. Tropifexor was highly protein bound (>99%) in human plasma across participants of all groups. The average unbound fractions (percent free) were 0.14% in participants with normal hepatic function and mild HI, which increased to 0.17% and 0.24% in participants with moderate and severe HI, respectively. Similar unbound drug exposure was noted in participants with mild HI and normal hepatic function. Participants with moderate HI (N = 8) had a 1.6-fold increase in unbound exposure (area under the plasma concentration-time curve from time 0 to infinity [AUCinf, u ]) and a 1.3-fold increase in maximal exposure (Cmax,u ) vs those with normal hepatic function (geometric mean ratio: AUCinf, u = 1.64 [90% CI, 1.25-2.16]; Cmax,u = 1.30 [0.96-1.76]). Participants with severe HI (N = 8) had a 1.6-fold increase in AUCinf, u (1.61 [1.04-2.49]) and comparable Cmax,u (1.02 [0.60-1.72]) compared to participants with normal hepatic function. Tropifexor was well tolerated. The relative insensitivity of tropifexor to HI offers the potential to treat patients with severe liver disease without dose adjustment. This article is protected by copyright. All rights reserved.

Objectives To explore the pharmacokinetics (PK) of oral and intravenous (iv) lefamulin after single and multiple doses, and the effect of food on bioavailability. Methods Lefamulin PK was examined in four studies. In Study 1, PK was assessed in patients with acute bacterial skin and skin structure infections who received repeated iv lefamulin q12h (150 mg). In Study 2, a four-period crossover study, healthy subjects received a single dose of oral lefamulin [immediate-release (IR) tablet, 1 × 600 mg] in a fasted and fed state, oral lefamulin (capsule, 3 × 200 mg) in a fasted state, and iv lefamulin in a fasted state. In Study 3, a three-period crossover study, healthy males received a single oral lefamulin dose (IR) in the following states: fasted, fasted followed by a high-calorie meal 1 h post-dose, and fed. Study 4 had two parts; in part A, healthy males received a single lefamulin dose (IR) in a fasted and fed state; in part B, subjects received repeated doses of lefamulin (IR, q12h) or placebo. Adverse events (AEs) were recorded in each study. Results Single and repeated dosing of iv and oral lefamulin resulted in comparable exposure. Intravenous and oral lefamulin (given fasted or with a meal 1 h post-dose) resulted in bioequivalence. Bioequivalence was not established between oral lefamulin in the fed state and iv or oral administration in the fasted state. All AEs were mild or moderate in severity, no serious AEs were reported, and no participant discontinued because of an AE. Conclusions The PK of lefamulin supports successful switch from iv to oral therapy; lefamulin was generally well tolerated.

These two Phase I, open-label, single-dose, randomized, crossoverstudies in 40 healthymale subjects investigated the pharmacokinetic and safety profiles of various formulations of the amprenavir prodrug GW433908 in the presence and absence of food compared with amprenavir capsules. GW433908 is a phosphate ester prodrug of the antiretroviral protease inhibitor amprenavir, with improved solubility over the parent molecule and a potential for reduced pill burden on current dosing regimens. The calcium salt of the prodrug, GW433908G, was selected for further investigation, as it appeared to offer the greatest potential for the development of new drug formulations. In the fasting state, (1) GW433908G tablet and suspension were bioequivalent in terms of both AUC and Cmax, and (2) GW433908G tablet and suspension were bioequivalent to amprenavir capsules for AUC; however, Cmax was lower with GW433908G. After a high-fat meal compared with fasting, (1) the bioavailability of GW433908G suspension was decreased by 20% and Cmax by 41%, and (2) for GW433908G tablets, there was no influence on AUC(12% lower Cmax). After a low-fat meal compared with fasting, (1) there was bioequivalence for GW433908G tablets, but (2) bioavailability was decreased by 23% for amprenavir capsules (Cmax was also lower, by 46%). Overall, for GW433908G and amprenavir capsules, food had a negligible influence on plasma concentration at 12 hours postdose (C12). Whether administered as tablets or suspension, GW433908G pharmacokinetics was only slightly affected by food. GW433908G tablets were well tolerated and delivered plasma amprenavir concentrations equivalent to the recommended therapeutic amprenavir dose but with fewer tablets. The possibility of a lower pill burden offered by GW433908 may be of clinical benefit in the treatment of HIV infection.

In order to choose a rational dose for GW 420867X, we first set a goal of therapy. We hypothesized that, for optimal antiretroviral activity, the trough free drug concentration should remain above the 90% effective concentration (EC 90 ) of human immunodeficiency virus type 1. We performed population pharmacokinetic analysis on three different doses of GW 420867X (50, 100, and 200 mg). Monte Carlo simulation was performed, assuming a log-normal distribution for 1,000 simulated subjects for each dose, and was repeated three times. The trough concentrations were divided by 76 to account for protein binding and for the difference between EC 50 and EC 90 . We then determined the fraction of the simulated population whose free drug trough concentrations would exceed an EC 90 over a broad range of values. The target attainment for all three doses exceeded 95% out to a starting EC 50 of 10 nM. For 16 viral isolates, the EC 50 range encountered for GW 420867X did not exceed 8 nM, implying that the three doses could not be differentiated by effect in a clinical trial in naive patients. This prediction was shown to be correct in a randomized, double-blind trial with 1 week of monotherapy with GW 420867X.

The aim of the current study was to characterize the pharmacokinetics of GW420867X, a new nonnucleoside reverse transcriptase inhibitor, using a single escalating dose protocol in healthy volunteers. Four dose levels were investigated in sequential order: 300, 600, 900, and 1200 mg, with a ratio of 4:1 subjects receiving active or placebo treatment, respectively. Following single-dose administration, GW420867X was readily absorbed with a median time to peak concentration of 3 to 5 hours. GW420867X plasma exposure (AUC) was dose proportional but variable within the 300 to 1200 mg dose range. Less than dose-proportional increases were observed for C max . The terminal elimination t 1/2 was 50 hours, which supports once-daily dosing in future studies. Plasma trough concentrations of GW420867X at 24 hours after dosing were many fold greater than the in vitro IC 50 HIV-1 HXB2 in MT4 cells. GW420867X was generally well tolerated following single-dose administration up to 900 mg; increased central nervous system-related adverse events were observed at higher doses. GW420867X had a favorable pharmacokinetic and safety profile that would enable this drug to be explored in future clinical studies with HIV-1 infected patients at doses that would provide appropriate safety and efficacy.

GW420867X is a nonnucleoside inhibitor of HIV-1 reverse transcriptase. The primary objective was to assess the safety of GW420867X in HIV-1-infected patients. The secondary objectives were to assess the effect of GW420867X on plasma HIV-1 RNA and viral genotype and phenotype and to examine the pharmacokinetics of GW420867X in HIV-1-infected patients.HIV-1-infected patients were randomized to GW420867X 50 mg/day, 100 mg/day, or 200 mg/day from days 1-28 (n = 15 per group). Lamivudine (3TC) plus zidovudine (ZDV) was added from days 8-28. A control group (n = 15) received GW420867X, 3TC, and ZDV placebos.Plasma HIV-1 RNA and CD4+ counts improved in the GW420867X groups at days 8 and 28. No significant development of drug resistance was detected. Median observed peak GW420867X concentration (C(max)) generally occurred at 2 hours. The area under the curve over the dosing interval (AUCtau)on day 14 increased less than proportionally to dose, suggesting there was increased clearance and/or decreased absorption. Mean trough GW420867X concentrations were many fold above the in vitro IC(50) in the presence of human serum proteins. Seven of 15 patients on 50 mg GW420867X, 8/15 on 100 mg GW420867X, 12/15 on 200 mg GW420867X, and 8/15 on placebo reported drug-related adverse events.GW420867X was well tolerated and has potent antiretroviral activity alone and in combination with 3TC plus ZDV.

Objective: To assess the pharmacokinetics, safety and tolerability of escalating oral doses of GW420867X, a non-nucleoside reverse transcriptase inhibitor, was investigated in healthy male volunteers in a randomized, double-blind placebo-controlled study. Methods: Study subjects were divided into four groups of 12 subjects (10, 50, 100 and 200-mg dose groups) with eight subjects from each group receiving active treatment and the remaining four matched placebo. Subjects were initially administered a single dose of GW420867X or placebo, and following a 24- to 28-day washout period, re-exposed to the same treatment for 14 consecutive days. Safety measurements including clinical laboratory evaluations, ECG and vital signs were performed before, during and after dosing. Results: Geometric mean GW420867X peak plasma concentrations (Cmax) following single oral doses of 10, 50, 100 and 200 mg were 160, 608, 1000 and 1662 ng/ml, respectively. Time to Cmax (tmax) increased from a median value of 1 h following the 10-mg dose, to 3 h after the 200-mg dose. Geometric mean plasma areas under the curves (AUC) were 4325 (10 mg), 17,862 (50 mg), 35,295 (100 mg) and 62,338 ng/ml per hour (200 mg) and were proportionally less than the increase in the administered dose. Apparent terminal elimination half-life (t1/2) was approximately 50 h. Following repeat dosing, accumulation ratios based on plasma AUC were: 3.0±1.0 (10 mg), 2.6±0.9 (50 mg), 1.8±0.3 (100 mg) and 1.9±0.8 (200 mg) after 14 days of dosing compared to the corresponding single dose. In general, oral clearance (CL/F) was greater after 14 days and greater with higher doses except for the 10-mg dose group. Steady-state CL/F was 2.2, 3.4, 4.2, and 5.1 l/h for 10, 50, 100, and 200 mg, respectively. Steady-state was generally achieved within 7–10 days. Comparison of single and repeat dosing with GW420867X showed that Cmax increased by a factor of between 1.4 to 1.8, after 14 days of daily dosing to 288 (10 mg), 1006 (50 mg), 1401 (100 mg) and 2613 (200 mg) ng/ml. These increases were proportionally less than the increase in the administered dose. GW420867X was well tolerated by subjects both after single and repeated dosing. Adverse effects reported by subjects on the active drug were similar to those receiving placebo. All episodes were rated as mild to moderate in severity and resolved spontaneously without further intervention. Conclusion: The pharmacokinetic findings of this study imply that systemic exposure to GW420867X decreases with increasing dose and displays time-variant pharmacokinetics, which suggests decreased absorption and/or increased clearance of GW420867X. The relatively long plasma half-life, of approximately 50 h, makes it suitable for once-daily dosing.

Recent data have proven that combination chemotherapy is a necessity for the long-term suppression of human immunodeficiency virus (HIV) (1, 5). Once one posits that combination chemotherapy is a necessity for successful HIV chemotherapy, the problem of determining optimal chemotherapeutic regimens is posed. Combination chemotherapy is an inherently difficult problem, as there is true between-patient variability in the pharmacokinetics of each agent in the combination. In addition, the pharmacokinetics of one agent may not provide clues to the handling of the second agent in the combination, particularly when the drugs are primarily cleared by different organs (e.g., clearance by the kidneys versus that by the liver). Finally, the drugs may interact in a synergistic, additive, or antagonistic fashion. Other variables which influence the observed outcome include the drug dose and schedule of administration. Determination of the drug interaction in a quantitative fashion is central to the delineation of optimal dosing regimens. Clearly, this is an important problem. The multiple regimens which need to be evaluated in a phase I-II trial of combination therapy means that many patients will be exposed to suboptimal regimens. Further, the cost in time and money of evaluating multiple regimens for dose and schedule is staggering. The ability to examine the impact of both dose and schedule in a quantitative fashion for combinations of agents can winnow the search for an optimal regimen to a manageable number, which can be appropriately studied in the clinical trial arena in the shortest time with the fewest number of patients. Recently, our group has published a paper detailing the interaction of abacavir plus amprenavir with regard to antiviral effect in a fully parametric fashion (2) by employing the equation of Greco et al (4). In this analysis, we demonstrated that the combination of abacavir and amprenavir was fully synergistic. In this study, our objective was to develop an approach which allows such data to be used to evaluate doses and schedules for combinations of agents. We have previously shown for an HIV protease inhibitor that time being greater than the 95% effective concentration (EC95) is the dynamically linked variable (G. L. Drusano, S. L. Preston, J. A. Bilello, B. Sadler, J. McDowell, W. Symonds, M. Rogers, S. LaFon, D. S. Stein, K. Muir, and A. Bye, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. A5, 1998), and we wished to explicitly assess the effect of schedule of administration of the protease inhibitor amprenavir on the antiviral effect of its combination with the potent nucleoside analogue abacavir. We examined the simulated effects of 300 mg of abacavir given orally every 12 h (q12h) in combination with either 1,200 mg q12h or 800 mg q8h of amprenavir.

Study Objectives. Study A: to determine the absolute bioavailability of a single 300-mg abacavir hemisulfate tablet. Study B: to determine the bioequivalence of two oral abacavir formulations (300-mg hemisulfate tablet, 100-mg succinate caplet), the effect of food on the bioavailability of the 300-mg hemisulfate tablet, and the bioavailability of the hemisulfate tablet relative to the hemisulfate solution. Design. Phase I, randomized, open-label, balanced two- (study A) and three-or four-period (study B), crossover studies. Setting. Two clinical research centers. Subjects. Six men infected with the human immunodeficiency virus (HIV), aged 27–39 years (study A), and 18 HIV-infected men and women, aged 21–50 years (study B). Interventions. In study A, all subjects received a single, oral 300-mg tablet of abacavir hemisulfate or a single, intravenous infusion of abacavir hemisulfate 150 mg over 60 minutes. In study B, all subjects received each of three single-dose treatments: three 100-mg abacavir succinate caplets in a fasted state, one 300-mg abacavir hemisulfate tablet in a fasted state, and one 300-mg abacavir hemisulfate tablet with a high-fat breakfast. Twelve subjects in study B also received a fourth treatment of abacavir hemisulfate 300 mg as an oral solution in a fasted state. Plasma samples collected for 24 hours (study A) or 12 hours (study B), and urine samples collected for 12 hours (study A) were analyzed by validated high-performance liquid chromatographic methods. Measurements and Main Results. Abacavir pharmacokinetic parameters were calculated using standard, noncompartmental methods. In study A, the geometric least square (GLS) mean absolute bioavailability of oral abacavir was 83% (range 65–107%). In study B, the hemisulfate tablet was bioequivalent to the succinate caplet, but its time to maximum concentration (Tmax) occurred 30 minutes earlier. Administration of the abacavir hemisulfate tablet with food had no effect on area under the curve from time zero to infinity (AUC0–∞), decreased maximum concentration (Cmax) by 26%, and delayed Tmax by 38 minutes. The relative bioavailability (GLS mean AUC0–∞ ratio) of the 300-mg abacavir hemisulfate tablet to solution was 101%, Cmax was 11% lower, and Tmax was unchanged. The most common drug-related adverse events associated with abacavir were nausea, vomiting, abdominal pain, and headache, all of which were mild. Conclusion. Based on our results, abacavir is safe and well tolerated and can be administered with or without meals.

The safety, antiretroviral activity, and pharmacokinetic profile of nelfinavir, a potent and specific inhibitor of human immunodeficiency virus (HIV) protease, were assessed in a small open-label phase I/II dose-ranging study in protease inhibitor-naive HIV-positive men. A total of 22 patients with baseline plasma HIV RNA ≥ 20,000 copies/mL and CD4+ counts between 200 and 500 cells/mm 3 were enrolled in the study. Of the 22 patients, 20 were evaluated for activity; 10 patients assigned to 771 mg/day base equivalent (300 mg three times daily) and 10 patients assigned to 1,026 mg/day base equivalent (600 mg twice daily) given monotherapy. A capsule formulation of nelfinavir was used. The initial study period was 28 days; patients showing a virologic response of 1 log 10 reduction were eligible for enrollment in an extension phase and addition of nucleoside analogues. A maximally tolerated dose of nelfinavir was not established. A dose-response relationship was observed for four (40%) patients in the 771-mg group and six (60%) patients in the 1,026-mg group experiencing a reduction from baseline in plasma HIV RNA of at least 1 log during the 28-day study. of these patients, five sustained the reduction in plasma HIV RNA beyond day 28 (2 patients receiving 771 mg/day and 3 patients receiving 1,026 mg/day). Median increases from baseline in CD4+ counts at day 28 were 216 cell/mm 3 and 86 cell/mm 3 in the 771-mg and 1,026-mg groups, respectively. After oral administration, median nelfinavir plasma concentrations on day 28 reached a maximum at 1 hour (2,966 ng/mL) in the 771-mg group and at 3 hours (3,157 ng/mL) in the 1,026-mg group. Data for 22 patients were included in the safety analysis; 12 patients (55%) reported at least one grade 2 or worse (moderate, severe, or very severe) adverse event. The most common grade 2 or worse adverse event was diarrhea, reported by two patients (20%) receiving 771 mg/day and seven patients (70%) receiving 1,026 mg/day; followed by nausea, flatulence, asthenia, and headache (each reported in 1 patient [10%] in the 771-mg group) and dizziness (reported in 1 patient [10%] receiving 1,026 mg/day). In the small subgroup (n = 6) who continued taking nelfinavir for longer periods (between 8 and 15 months), virologic responses were sustained in the majority of patients with good tolerability. Nelfinavir is an active HIV-protease inhibitor with favorable pharmacokinetics, good tolerability, and sustained antiviral effects. Results of this early phase I/II dose-ranging study provided data for the safety and antiretroviral activity of nelfinavir and led to the selection of higher doses for phase II/III trials to further optimize virologic and immunologic responses.

Atherosclerosis is a leading cause of morbidity and mortality worldwide. Statins are established as first choice drugs for the management of hyperlipidaemia and cardiovascular risk. However, a residual cardiovascular risk, partially attributable to lipids, remains even after statin treatment. This risk appears to be associated with both high-density lipoprotein cholesterol and triglyceride lipid fractions. Several novel therapeutic approaches have been proposed to reduce lipid levels. Microsomal transfer protein (MTP) is involved in the assembly of very-low-density lipoprotein and chylomicron lipoprotein particles in the liver and the gut, respectively. In the preclinical setting, various agents that affect activity of MTP have shown that inhibition can result in profound reductions in blood triglycerides and cholesterol. Similarly, evidence of efficacy using the target has been confirmed in man with small molecule inhibitors and antisense oligonucleotides. Unfortunately, despite their efficacy in reducing lipids, the clinical utility of small molecule inhibitors has been restricted by their potential to induce hepatic steatosis. Continuing attempts to utilise this clinical target (to decrease cholesterol, triglycerides and weight) have involved the use of lower doses or non-systemically absorbed MTP inhibitors.

(S)-2-ethyl-7-fluoro-3-oxo-3, 4-dihydro-2H-quinoxaline-carboxylic acid isopropylester (GW420867X) inhibits HIV-1 reverse transcriptase and could be used for the treatment of HIV infection. This study quantified the movement of [ 14 C]GW420867X into the CNS by means of a guinea-pig brain perfusion technique. Results indicated that [ 14 C]GW420867X can enter the brain (K in ; 38.4 ± 7.7 μl min -1 g -1 ) and cerebrospinal fluid (CSF; K in : 1.2 ± 0.1 μl min -1 g -1 ). Self-inhibition studies also suggested the presence of a saturable transport system for [ 14 C]GW420867X at the blood-brain barrier (BBB). Thus [ 14 C]GW420867X can enter the brain via the BBB and, compared with the blood-CSF barrier, this route is the predominant pathway for the brain entry of this drug. This would suggest that GW420867X is a promising drug for the treatment of HIV infection within the brain.

Cajin, the toxic principle extracted from the female abdomen and eggs of the aposematic moth Arctia caja, was investigated by means of column chromatography, and its effects on tissues were studied by electrophysiology and electron microscopy. Partial purification suggests that one fraction is a polypeptide with a molecular weight (mol. wt) of about 1000. It is lethal to both mammals and insects if injected into the body, but inactive via the oral route. Crude extracts applied to insect muscles produce a slow contracture which lasts for over 1 1 2 hr and is irreversible. Fine structural analyses of insect tissues after 5 min toxin treatment reveal disruption of both internal membranous systems and mitochondria. Unlike many other toxins, cajin appears to act by stimulating the muscle directly. The results of experiments involving Ca2+-free solutions plus cajin suggest that it may increase the permeability of the muscle membrane to Ca2+, perhaps behaving as a calcium ionophore, or may act as an analogue of an insect neurotransmitter.

The ionophore A-23187 at a concentration of 10 −6 M stimulates fluid secretion, increases calcium influx and efflux, and alters the trensepithelial potential in the isolated fly salivary gland but causes a fall in cyclic AMP concentration. In this regard, it mimics all the effects of 5-hydroxytryptamine of this tissue, except that 5-hydroxytryptamine causes a rise in cyclic AMP concentration. These data in conjunction with past evidence indicate that calcium, as well as cyclic AMP, is a second messenger in the action of 5-hydroxytryptamine upon fly salivary gland secretion.

1. The role of calcium in the potential and secretory responses of isolated salivary glands of Calliphora to 5-HT and cyclic AMP has been studied. 2. Secretion induced by 5-HT was reversibly inhibited by removal of calcium from the bathing medium. 3. The chloride-dependent depolarization of the apical membrane produced by 5-HT was calcium dependent whereas the potential response to cyclic AMP was little effected. 4. Strontium and barium effectively substituted for calcium. 5. Manganese replaced calcium at the onset of secretory and potential responses but these responses were maintained when manganese was removed. 6. Lanthanum did not substitute for calcium in secretory responses but did inhibit the secretory and potential responses to calcium in calcium-depleted glands. 7. The rate of secretion in a low-chloride medium produced by cyclic AMP was significantly lower than that induced by 5-HT, but there was little difference in normal saline. 8. A model for the mode of action of 5-HT is proposed in which calcium acts as an intracellular intermediary controlling chloride movements whilst cyclic AMP controls a potassium pump.

Fluid transport across epithelia plays an important role not only in osmoregulation, but also in digestion. In many cases, the rate of fluid movement is carefully regulated by hormonal or nervous stimuli. Although the nature of the hormones and nerves responsible for this regulation have been characterized, not much is known about how these various stimuli bring about the very large changes in fluid flow which are seen during stimulation of such epithelia as the pancreas or mammalian salivary gland. One reason for our ignorance about how epithelia are controlled stems from the fact that we still know very little about how ions and water are transported across most epithelia. In order to unravel the control mechanisms within epithelial cells, it is first necessary to understand the basic features of the transport mechanisms. Such essential information has been difficult to obtain for most vertebrate epithelia because of their structural complexity. Such problems are greatly reduced in various insect epithelia such as the salivary glands of adult blowflies. These insect salivary glands not only have a relatively simple structure (1) but they are particularly amenable to in vitro studies which have provided insights into both the mechanism and the control of secretion.

Fluid secretion by isolated salivary glands was stimulated by elevating the external potassium concentration. The stimulatory effect of potassium was dependent on external calcium and was potentiated by a subthreshold dose of 5-hydroxytryptamine (5-HT). During the action of 120 mM potassium there was a large calcium dependent decrease in transepithelial resistance similar to that produced with 5-HT at normal potassium concentrations. These results on Calliphora salivary glands are compared with other cases where cells are activated by high potassium. In most cases, the effect of high potassium is dependent upon calcium, suggesting that the latter plays a primary role in cell activation.

Publisher Summary This chapter reviews the progress, which has been made in understanding how one particular cell system, the salivary gland of an insect, receives a chemical stimulus and translates the information into a change in activity. Comparison of the mode of action of 5-HT in Calliphora salivary glands with that of many other hormones reveals numerous similarities. External chemical stimuli responsible for regulating cellular activity may thus share a common mode of action. The various differences which are apparent today may represent modification of this basic mechanism developed during the course of evolution to adapt cells systems to new environments. The main feature of this control system concerns the transduction of hormonal information arriving at the cell into internal chemical signals, which can be recognized by the various effector mechanisms responsible for carrying out the actions of the hormone. In the original cyclic AMP-hypothesis, the hormonal signal input was transduced into cyclic AMP through an activation of the membrane-bound enzyme adenyl cyclase and cyclic AMP alone then mediated all further cellular events. The current hypothesis proposed by Rasmussen (1970) and verified in these salivary gland studies is that the conformational changes induced in the membrane by successful hormone-receptor interactions can be transduced into more than one secondary message.

The sites of the transepithelial potential changes produced by 5-HT and cyclic AMP in the salivary gland of the blowfly (Calliphora) have been investigated. Microelectrodes recorded a two-step potential profile across this epithelium. The cell was negative to both the bathing medium and the saliva. The basal membrane potential was 44·0 ± 0·2 mV and was affected very little by application of either 5-HT or cyclic AMP. The apical membrane was depolarized by 5-HT and in contrast was hyperpolarized by cyclic AMP. On the basis of these results it is concluded that 5-HT has two actions: (a) to stimulate a potassium pump on the apical membrane using cyclic AMP as an intermediary, (b) to increase chloride movement by a mechanism not directly involving cyclic AMP.

The role of cyclic AMP in mediating the action of 5-HT on salivary glands has been studied by measuring transepithelial potentials. The lumen of unstimulated glands is 4 mV positive but becomes 12 mV negative after treatment with 5-HT (10−8M). Both the potential and the secretory responses to 5-HT are dose-dependent over the same concentration range. The electrical response of salivary glands to cyclic AMP is qualitatively different to that of 5-HT; instead of going negative the potential goes more positive. An increase in positive potential is also observed after treatment with theophylline (10−2M), or when glands are stimulated with 5-HT in a chloride-free saline. These results are consistent with the idea that 5-HT has two actions. One is to stimulate the enzyme adenyl cyclase to synthesize cyclic AMP, which, in turn, stimulates cation transport. The other is to increase anion transport by a mechanism which is independent of cyclic AMP.

Rate of fluid secretion by the salivary glands of the blowfly Calliphora is regulated by 5-hydroxytryptamine (5-HT) working in conjunction with cyclic AMP. Although cyclic AMP can exactly mimic the acceleration of fluid secretion produced by 5-HT, the underlying electrical events are completely different. Transepithelial potentials were measured by a liquid paraffin-gap technique which permits continuous potential recordings during rapid changes of the bathing medium. The potential of the lumen of unstimulated glands is + 5 mV with respect to the bathing medium but becomes — 10 to 20 mV after applying 5-HT. After stimulation with cyclic AMP, however, the luminal potential becomes more positive (+ 30 to 40 mV). A similar effect is obtained with theophylline or when glands are treated with 5-HT in the presence of an impermeant anion such as isethionate. These observations suggest that in addition to stimulating the synthesis of cyclic AMP, 5-HT may also act directly to increase anion movement. Cyclic AMP appears to stimulate cation transport, which explains the increase in positive potential obtained when this compound (or theophylline) is applied in the absence of 5-HT.

Cyclic nucleotide has been found to play an important role in the activation of many different cells by specific stimuli. It has been found to be involved in the phenomena of enzyme induction in bacteria of aggregation in the cellular slime mold Dictyostelium discoideum of the action of many hormones, and in a wide variety of secretory processes including the release of synaptic transmitters. One need only contrast the function of these enzymes in liver mitochondria to those in insect flight muscle to make the point. In the liver, they participate, in times of fasting, in the conversion of amino acids to cytoplasmic carbohydrate precursors, of fatty acids to ketone bodies, and to cytoplasmic reducing equivalents. In the case of secretion and peptide hormone action, the requirement for calcium and the activation of membrane-bound adenyl cyclase both appear in many of the same systems. An important aspect of this relationship involves the effects of cyclic AMP and first messenger on calcium uptake and release by particular cells.

The action of 5-hydroxytryptamine (5-HT) on an insect salivary gland was associated with a rise in adenosine-3′:5′-cyclic monophosphate (cAMP) concentration and an increase in calcium uptake. An increase in secretion induced either by 5-HT or exogenous cAMP required extracellular calcium. Both 5-HT and exogenous cAMP increased 45 Ca efflux from previously labeled glands, but only 5-HT caused an increase in calcium uptake. The transepithelial potential in this tissue became more negative after addition of 5-HT, but more positive after addition of cAMP. 5-HT and cAMP induced a more negative potential when calcium was removed from the medium. It was concluded that both calcium and cAMP serve as intracellular messengers when 5-HT acts upon fly salivary gland.