Water and enzyme secretion are tightly coupled in pancreatic secretion stimulated by food or CCK-58 but not by CCK-8

Pancreatic secretion of protein, water, chloride, and bicarbonate under basal conditions and in response to intravenous and intraduodenal stimuli were studied in awake rats fully recovered from surgery. During the basal phase of pancreatic secretion, protein output and water output were weakly correlated or uncorrelated, consistent with separate regulation and distinct cellular origin of enzyme (acinar cells) and water (duct cells), referred to as the two-component paradigm of pancreatic secretion. When pancreatic secretion was stimulated physiologically, water and protein output abruptly became strongly and significantly correlated, suggesting that protein secretion and water secretion are tightly coupled or that protein secretion is dependent on water secretion. The apparent function of this coupling is to resist or prevent increases in protein concentration as protein output increases. This pattern of secretion was reproduced by intravenous infusion of the CCK-58 form of cholecystokinin, which strongly stimulates pancreatic water and chloride secretion, but not by CCK-8, which only weakly stimulates water and chloride secretion in a non-dose-dependent manner. The remarkably tight association of water and protein secretion in food-stimulated and CCK-58-stimulated pancreatic secretion is consistent with a single cell type as the origin of both water and enzyme secretion, i.e., the acinar cell, and is not consistent with the two-component paradigm of pancreatic secretion. Because CCK-58 is the only detectable endocrine form of cholecystokinin in the rat and its bioactivity pattern is markedly and qualitatively different from CCK-8, actions previously recorded for CCK-8 should be reexamined.     

Immunosuppression induced by central action of morphine is not blocked by mifepristone (RU 486)

Morphine causes immunosuppression by binding to opioid receptors on immune cells, or indirectly by acting on receptors in the brain. However, morphine exact mechanism of action has not been elucidated. In the present study, we investigated the role of glucocorticoids in morphine-mediated immunosuppression after acute action in the rat mesencephalon periaqueductal gray (PAG). Natural killer (NK) cell activity and T cell proliferation were used to evaluate potential indirect mechanisms of morphine action. Microinjection of morphine in the ventral-caudal aspect of the PAG significantly (p < 0.01) suppressed splenic NK cell cytotoxic activity (32% reduction), and antiTCR-, IL-2-, antiTCR + IL-2, and Con A-induced thymic (30% to 50% reduction) and splenic (35% to 70% reduction) lymphocyte proliferation compared with PAG-injected saline control animals. The glucocorticoid receptor antagonist mifepristone (RU 486) did not block the immunosuppressive effects of morphine, suggesting that such effects are independent of activation of the hypothalamic-pituitary-adrenal axis.     

Modulation of ethanol-intake by morphine: evidence for a central site of action

Previous studies have shown that subcutaneous administration of low doses of morphine increase, while subcutaneous naloxone decreases, ethanol-intake in rats. However, the site of action of morphine modulation of ethanol-intake remains unclear. In an attempt to elucidate this issue, seven graded doses of morphine were given intracerebroventricularly to rats 15 min prior to an opportunity to consume water and sweetened alcoholic beverage for 2 hr. Two lower doses of intracerebroventricular morphine (1, 3 micrograms) reliably increased ethanol-intake, while higher doses (3-30 micrograms) decreased intake of water. Preference ratios (ethanol-intake divided by total fluid-intake) were reliably increased by morphine doses of 1 microgram and higher. The present data provide support for a central site of morphine modulation of ethanol-intake.     

Modulation of CCK-8-evoked intracellular Ca2+ waves by hydrogen peroxide in mouse pancreatic acinar cells.

In the present study we have employed single cell imaging analysis to monitor the propagation of cholecystokinin-evoked Ca(2+) waves in mouse pancreatic acinar cells. Stimulation of cells with 1 nM CCK-8 led to an initial Ca(2+) release at the luminal cell pole and subsequent spreading of the Ca(2+) signal towards the basolateral membrane in the form of a Ca(2+) wave. Inhibition of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) activity by 1 microM thapsigargin, preincubation in the presence of 100 microM H(2)O(2) or inhibition of PKC with either 5 microM Ro31-8220 or 3 microM GF-109203-X all led to a faster propagation of CCK-8-induced Ca(2+) signals. The propagation of CCK-8-evoked Ca(2+) signals was slowed down by activation of PKC with 1 microM PMA, and preincubation of cells in the presence of H(2)O(2) counteracted the effect of PKC inhibition. The protonophore FCCP (100 nM) and the inhibitor of the mitochondrial Ca(2+)-uniporter Ru360 (10 microM) led to an increase in the propagation rate of CCK-8-evoked Ca(2+) waves. Finally, depolymerisation of actin cytoskeleton with cytochalasin D (10 microM) led to a faster propagation of CCK-8-evoked Ca(2+) signals. Stabilization of actin cytoskeleton with jasplakinolide (10 microM) did not induce significant changes on CCK-8-evoked Ca(2+) waves. Preincubation of cells in the presence of H(2)O(2) counteracted the effect of cytochalasin D on CCK-8-evoked Ca(2+) wave propagation. Our results suggest that spreading of cytosolic Ca(2+) waves evoked by CCK-8 can be modulated by low levels of oxidants acting on multiple Ca(2+)-handling mechanisms.     

Opioids and central baroreflex control: a site of action in the nucleus tractus solitarius

These studies investigated whether the nucleus of the tractus solitarius (NTS) is a central site where opioids modulate baroreceptor reflexes. Microinjections into the NTS of [D-Ala2,MePhe4, Gly-ol5]enkephalin (DAGO) significantly reduced reflex-mediated depressor responses evoked by electrical stimulation of the aortic nerve. Subsequent NTS injections of naloxone restored baroreflexes to control levels. These results demonstrate that the NTS is a central site where exogenously administered opioids can modulate baroreceptor reflexes. NTS injections of naloxone had no effect on baroreflex function, suggesting that tonic activation of opioid receptors at this site plays little or no role in central baroreflex control.     

Acute and long-lasting effects of peripheral injection of caerulein and CCK-8 on the central GABAergic system in mice

Acute and long-lasting effects of peripheral injection of caerulein (CLN) and cholecystokinin octapeptide (CCK-8) on the gamma-aminobutylic acid (GABA) content and the GABA accumulation by aminooxyacetic acid (AOAA) in the discrete brain regions of mice were examined. The content and accumulation of GABA in the striatum, hypothalamus, and frontal cortex was measured with high performance liquid chromatography with electrochemical detection (HPLC-ECD). The GABA content slightly decreased in the striatum 60 min after CLN and CCK-8 were administered, whereas it slightly increased in the hypothalamus and frontal cortex. Moreover, with CLN and CCK-8, the GABA accumulation after AOAA treatment decreased in the striatum and hypothalamus 30 min after injection. Meanwhile, when administering CLN, the GABA content as well as the GABA accumulation after AOAA treatment increased in the striatum and frontal cortex 1 day after injection, and continued to increase the second and third day in the striatum. These results showed that peripheral injection of CLN and CCK-8 had effects on the central GABAergic system with local specific actions, and also the long-lasting and time-dependent biphasic effects of CLN.     

Presence and possible site of action of secretin in the rat pituitary and hypothalamus

Secretin-like immunoreactivity was detected in extracts of several rat brain structures by radioimmunoassay, most notably in the pituitary, hypothalamus, pineal and septum. Its localization to these structures suggested that it might play a role in neuroendocrine events similar to its structural homolog vasoactive intestinal peptide. Dose-related stimulations (MED, 10(-7) M) of prolactin (PRL) release were observed after incubation of synthetic secretin with dispersed, cultured pituitary cells from male and ovariectomized (OVX) female rats. In OVX females, i.v. infusion of a high dose of secretin (10 micrograms) resulted in a significant elevation of PRL levels. Doses of secretin as low as 0.1 micrograms when administered into the third cerebroventricle were capable of significantly inhibiting PRL release in both males and OVX females, suggesting an ultrashort-loop, negative feedback of secretin. Secretin can now be added to the growing list of putative PRL-releasing agents.     

In vivo release of CCK-8 from the dorsal horn of the rat: Inhibition by DAGOL

There is evidence that CCK-8 may interact with opioids and that both systems are probably implicated in pain modulation. In order to elucidate this relationship we sought to examine factors governing the movement of CCK-8 from the spinal cord into the extracellular space. We report that CCK-8 like immunoreactivity, as measured by RIA, is released from the spinal cord of the rat in vivo, following potassium stimulation and by direct activation of high threshold peripheral afferents by stimulation of the sciatic nerve. Also, we show that CCK-8 release is inhibited by the μ-selective opioid receptor agonist DAGOL. Naloxone totally reversed the effect produced by DAGOL, implying an opiate mediated mechanism.     

Killing of targets by CD8 T cells in the mouse spleen follows the law of mass action

It has been difficult to correlate the quality of CD8 T cell responses with protection against viral infections. To investigate the relationship between efficacy and magnitude of T cell responses, we quantify the rate at which individual CD8 effector and memory T cells kill target cells in the mouse spleen. Using mathematical modeling, we analyze recent data on the loss of target cells pulsed with three different peptides from the mouse lymphocytic choriomeningitis virus (LCMV) in mouse spleens with varying numbers of epitope-specific CD8 T cells. We find that the killing of targets follows the law of mass-action, i.e., the death rate of individual target cells remains proportional to the frequency (or the total number) of specific CD8 T cells in the spleen despite the fact that effector cell densities and effector to target ratios vary about a 1000-fold. The killing rate of LCMV-specific CD8 T cells is largely independent of T cell specificity and differentiation stage. Our results thus allow one to calculate the critical T cell concentration at which growth of a virus with a given replication rate can be prevented from the start of infection by memory CD8 T cell response.     

Alphafetoprotein is not a component of the 8 S estradiol receptor from the immature mouse uterus or brain.

The hypothesis that alphafetoprotein (AFP) could be a consitituent of the estradiol receptor in the uterus and brain of the immature mouse has been tested. Cytosols prepared from 2 week-old mice were depleted of AFP by immunoadsorption with a Sepharose-coupled anti-mouse AFP anti-serum. Aliquots of these cytosols containing nondetectable levels of AFP by RIA were then brought to 0.4 M KCl to convert 8 S estradiol receptors to the 4-5 S forms, reacted either with anti-AFP or with a control anti-IgG immunoadsorbent, labeled with 3H-estradiol, and centrifuged on glycerol gradients. There was no, or very little, loss of estradiol receptors in the experimental as compared to the control cytosols indicating that AFP is not a constituent of the 8 S estradiol receptor.     

The possible involvement of GABA mechanisms in the action of benzodiazepines on central catecholamine neurons.

With the use of quantitative microspectrofluorometry, it has been shown that diazepam (10 mg/kg) and chlordiazepoxide (10 mg/kg) reduce DA turnover in the tuberculum olfactorium, nuc. accumbens, the DA islands of the entorhinal cortex, and caput of nuc. caudatus, whereas DA turnover is increased in the lateral external layer of the median eminence after 10 mg/kg of diazepam. It is of considerable interest that with a dose of 1 mg/kg of diazepam a reduction of DA turnover can still be observed in the tuberculum olfactorium and nuc. accumbens but not in the nuc. caudatus, due to a high variability of the response in this area. A similar trend is also found with chlordiazepoxide. Thus, changes in limbic DA turnover are observed in doses close to the minimal effective dose (0.6 mg/kg) needed to release punished behavior and to cause anticonvulsive effects, and may therefore be related to these actions of diazepam. For various reasons it is speculated that an increased GABA receptor activity on the DA cell bodies and their dendrites could mainly be involved in causing the reduction of DA turnover observed after benzodiazepines by diminishing the firing rate in the ascending DA pathways, particularly the mesolimbic DA pathways. Evidence for a change of GABA turnover by diazepam has also been found. It is also suggested that the reduction of cortical NE turnover found after benzodiazepines can partly involve an increased GABA receptor activity on the locus ceruleus cells, although the activation of E receptors on these cells cannot be excluded. These effects on locus ceruleus may be partly responsible for the sedation found after benzodiazepines. Diazepam (1 mg/kg) mimics both clonidine and GABA-ergic drugs in reducing blood pressure and slowing respiration rate, but the effects are blocked by picrotoxin but not by piperoxane, an E receptor-blocking agent. In agreement with the view that blockade of the stress-induced increases of NE turnover by benzodiazepines may be related to their antianxiety actions, it was found that the increase in NE turnover elicited by yohimbine, a drug that causes anxiety in man, is blocked by diazepam.     

Nonalgebraic interaction between pressor and depressor reflexes in dogs: evidence for a central site of action

In a previous study (Kendrick, JE and Matson, G 1979, Amer J Physiol 327:H662-H667) we demonstrated that the vascular responses in dogs to electrical stimulation of aortic nerve (AN) pressor and carotid sinus nerve (CSN) depressor afferents did not sum algebraically. We suggest this results from a reflex interaction which occurs in the central nervous system. The present study extends earlier studies by recording sympathetic vasomotor in chloralose-anesthetized dogs. Stimulation of the CSN reduced sympathetic activity by 51 +/- 20 (SD)%. AN stimulation (2 Hz) caused a 17 +/- 12% increase in sympathetic activity. Combined stimulation of the ipsilateral CSN and AN caused 0 +/- 28% change rather than a 34% decrease expected by an additive interaction. The interaction recorded in this study from the sympathetic outflow is therefore consistent with the previously reported vascular responses (cited above) and implicates central nervous site(s) of action. A conditioning stimulus train to CSN inhibited sympathetic discharges to AN test stimuli. This inhibition was prevented by pairing an AN stimulus with the CSN stimulus train. The AN pressor reflexes act in part by increasing sympathetic activity and in part by suppressing the baroreflexes.     

Supramaximal CCK-58 does not induce pancreatitis in the rat: role of pancreatic water secretion

In contrast to supramaximal CCK-8 or caerulein, acute or prolonged supraphysiological levels of endogenous CCK-58 do not cause pancreatitis. Compared with CCK-8, CCK-58 is a much stronger stimulant of pancreatic chloride and water secretion, equivalent to maximally effective secretin, but with a chloride-to-bicarbonate ratio characteristic of acinar fluid. Because supraphysiological endogenous CCK does not cause pancreatitis and because coadministration of secretin ameliorated caerulein- or CCK-8-induced pancreatitis, coincident with restoring pancreatic water secretion, we hypothesized that supramaximal CCK-58 would not induce pancreatitis. Conscious rats were infused intravenously with 2 or 4 nmol x kg(-1) x h(-1) of CCK-8 or synthetic rat CCK-58 for 6 h, and pancreases were examined for morphological and biochemical indexes of acute pancreatitis. A second group was treated as above while monitoring pancreatic protein and water secretion. CCK-8 at 2 nmol x kg(-1) x h(-1) caused severe edematous pancreatitis as evidenced by morphological and biochemical criteria. CCK-58 at this dose had minimal or no effect on these indexes. CCK-58 at 4 nmol x kg(-1) x h(-1) increased some indexes of pancreatic damage but less than either the 2 or 4 nmol x kg(-1) x h(-1) dose of CCK-8. Pancreatic water and protein secretion were nearly or completely abolished within 3 h of onset of CCK-8 infusion, whereas water and protein secretion were maintained near basal levels in CCK-58-treated rats. We hypothesize that supramaximal CCK-58 does not induce pancreatitis because it maintains pancreatic acinar chloride and water secretion, which are essential for exocytosis of activated zymogens. We conclude that CCK-58 may be a valuable tool for investigating events that trigger pancreatitis.     

Mechanism of action of cysteine proteinases: oxyanion binding site is not essential in the hydrolysis of specific substrates

To study the possible stabilization of the oxyanion of the tetrahedral intermediate formed in the course of the catalyses by cysteine proteinases, papain, chymopapain, papaya peptidase A, and ficin, we synthesized N-(benzyloxycarbonyl)phenylalanylthioglycine O-ethyl ester and compared its hydrolysis with that of the corresponding oxygen ester, a highly specific substrate of the above enzymes. It was found that the substitution of sulfur for the carbonyl oxygen hardly affected the second-order rate constant of acylation and diminished catalytic activity by about 1 order of magnitude in deacylation. These results contrast with those obtained with serine proteinases [Asbóth, B., & Polgár, L. (1983) Biochemistry 22, 117-122], where the hydrolysis of thiono esters could not be detected. From the results the following conclusions can be drawn. Stabilization of the tetrahedral intermediate at an oxyanion binding site is not essential with cysteine proteinases. Therefore, and because of the lack of general base catalysis, cysteine proteinases have a less constrained transition-state structure than serine proteinases.     

Expression of the mouse PR domain protein Prdm8 in the developing central nervous system

It was first shown in the PR (PRDI-BF1 and RIZ homology) domain family proteins that the PR domain has homology to the SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) domain, a catalytic domain of the histone lysine methyltransferases. Recently, there are many reports that the PR domain proteins have important roles in development and/or cell differentiation. In this report, we show the expression patterns of one of the mouse PR domain proteins, Prdm8, in the developing central nervous system. In the developing retina, Prdm8 expression was detected in postmitotic neurons in the inner nuclear layer and the ganglion cell layer, and its expression became restricted predominantly to the rod bipolar cells when retinogenesis was completed. In the developing spinal cord, Prdm8 was expressed first in the progenitor populations of ventral interneurons and motor neurons, and later in a subpopulation of interneurons. In the developing brain, Prdm8 expression was observed in postmitotic neurons in the intermediate zone and the cortical plate. In the postnatal brain, Prdm8 was expressed mainly in layer 4 neurons of the cerebral cortex. These results show that Prdm8 expression is tightly regulated in a spatio-temporal manner during neural development and mainly restricted to postmitotic neurons, except in the spinal cord.     

Airway hyperresponsiveness induced by cationic proteins in vivo: site of action

Major basic protein and other native cationic proteins increase airway hyperresponsiveness when administered to the luminal surface of the airways in vitro. To determine whether the same applies in vivo, we assessed airway responsiveness in rats challenged with both aerosolized and intravenously infused methacholine. We partitioned total lung resistance into its airway and tissue components using the alveolar capsule technique. Neither poly-l-lysine nor major basic protein altered baseline mechanics or its dependence on positive end-expiratory pressures ranging from 1 to 13 cmH(2)O. When methacholine was administered to the lungs as an aerosol, both cationic proteins increased responsiveness as measured by airway resistance, tissue resistance, and tissue elastance. However, responsiveness of all three parameters was unchanged when the methacholine was infused. Together, these findings suggest that cationic proteins alter airway responsiveness in vivo by an effect that is apparently limited to the bronchial epithelium.