Long-term salt loading impairs pituitary responsiveness to ACTH secretagogues and stress in rats

Male Wistar rats were allowed to drink tap water ad lib (W), 2% saline (S) or 2% saline containing dexamethasone (S + D, 1 mg/l) for 7 days. On the 8th day rats were subjected to a 3-min ether stress. Plasma ACTH, corticosterone and prolactin concentrations were determined before and after ether exposure. Prestress concentrations of plasma ACTH were low and did not vary among the three groups. In response to ether stress W rats exhibited twice as high plasma ACTH concentrations as did S rats. Rats of the S + D group exhibited a small but statistically significant ACTH response. Plasma corticosterone concentration in S rats was increased while in S + D rats was significantly decreased under resting conditions compared to that in W rats. Ether stress caused large increases in plasma corticosterone concentrations in W and S rats while a small but statistically significant increase was observed in S + D rats. Prolactin responses to ether were smaller in groups S and S + D than in group W. To test whether the decreased ACTH response to ether exposure was a result of a decreased sensitivity of corticotrope cells to corticotropin releasing factor (CRF)-41 or arginine vasopressin (AVP), adenohypophysial fragments from W, S and S + D rats were incubated in the presence of different doses of CRF-41 or AVP. Pituitary fragments obtained from W rats secreted larger amounts of ACTH than did pituitaries from S rats in response to either CRF-41 or AVP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic peptide does not affect corticotropin-releasing factor-, arginine vasopressin- and angiotensin II-induced adrenocorticotropic hormone release in vivo or in vitro

The effect of synthetic atrial natriuretic peptide (ANP) was examined on the in vivo and in vitro release of ACTH. Intravenous ANP (4 micrograms/kg body weight) administration did not affect the corticotropin releasing factor (CRF, 4 micrograms/kg body weight)-, arginine vasopressin (AVP, 2 micrograms/kg body weight)- and angiotensin II (A II, 4 micrograms/kg body weight)-induced ACTH release in unanesthetized freely moving rats. ANP did not inhibit the basal, CRF- and AVP-induced release of ACTH in pituitary cell cultures. ANP did not affect the CRF- and AVP-induced plasma corticosterone elevation, while it attenuated the AVP-induced corticosterone elevation. These results indicate that ANP does not affect the ACTH release at the pituitary level in vivo and in vitro.     

Effects of experimental hypothyroidism on the development of the hypothalamo-pituitary-adrenal axis in the rat

Hypothyroid pups were obtained by adding methimazole in the mother's drinking water from day 15 of gestation and sacrificed at 4, 8 or 15 days. Circulating corticosterone decreased at all ages, while CBG concentrations diminished at day 4, increased at day 8 and did not change at day 15 in hypothyroid rats. As opposed to controls, plasma ACTH concentrations decreased steadily with age while there was an accumulation of ACTH in the anterior pituitary of hypothyroid 15-day-old rats. Anterior pituitary POMC contents were unaffected by the treatment. In the hypothalamic PVN, CRF mRNA levels in the total population of CRF-synthesizing cells and in the CRF+/AVP+ subpopulation were below those of controls whatever the age considered while AVP mRNA in the CRF+/AVP+ subpopulation did not change at day 4 and decreased at day 8 and 15 in hypothyroid animals. Both the number of cell bodies expressing detectable levels of CRF mRNA and the percentage of CRF and AVP colocalization decreased at day 4 and were unchanged thereafter. CRF and AVP immunoreactivity in the zona externa of the median eminence increased with age but was not affected by methimazole treatment. The concentration of AVP mRNA in the magnocellular cell bodies of the PVN and the SON as well as AVP immunoreactivity in the zona interna of the median eminence were not changed by the treatment at days 4 and 8. In hypothyroid 15-day-old rats, SON AVP mRNA increased, AVP immunoreactivity decreased while plasma osmolality was enhanced. In conclusion, our data demonstrate that experimental hypothyroidism impairs specifically the maturation of hypothalamic parvocellular CRF and AVP gene expression during the stress hyporesponsive period. These observations suggest that the physiological peak in plasma thyroxine concentrations that occur between day 8-12 may participate in the maturation of hypothalamic CRF- and AVP-synthesizing cells.     

In vivo potentiation of corticotropin releasing factor activity by vasopressin analogues

The ability of the neurohypophyseal hormones and related synthetic peptides to potentiate the action of synthetic ovine corticotropin releasing factor (CRF-41) was examined using male rats whose endogenous CRF release was blocked with chlorpromazine, morphine and nembutal. CRF potency was clearly related to the pressor activity of the analogues. However, the threshold dose for eliciting a significant corticosterone response with the neurohypophyseal hormones was greater than with CRF-41. When arginine vasopressin (AVP) was coadministered with CRF-41 at subthreshold doses of both peptides, a significant corticosterone response was observed. When the neurohypophyseal hormone analogues were compared with regard to intrinsic CRF bioactivity, it was observed that an L-basic residue in sequence position 8 is necessary for high intrinsic activity. With one exception, l-Deamino-(9-D-Ala) arginine vasopressin, the ability to potentiate the effect of CRF-41 was related to the intrinsic CRF potency of the analogues. These results support previous reports of in vitro potentiation of CRF-41 by AVP and point out the complexity of CRF-neurohypophyseal hormone interaction in vivo.     

Fos protein expression in mouse hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei upon osmotic stimulus: colocalization with vasopressin, oxytocin, and tyrosine hydroxylase

The quantity and topography of activated vasopressin (AVP), oxytocin (OXY), and tyrosine hydroxylase (TH) neurons were studied immunohistochemically in the anterior, middle, and posterior portions of the PVN and SON in mice 60 min after a single injection of hypertonic saline (HS, 400 microl 1.5M, i.p.). Fos-neuropeptide double-stainings revealed: (1) Fos expression in each portion of the PVN and SON; (2) maximal number of Fos-AVP (79 cells) and Fos-OXY (50 cells) double-labelings in the middle portion of the PVN; (3) low number of Fos-TH perikarya in the PVN and their lack in the SON; (4) similar incidence (around 50%) of Fos-AVP and Fos-OXY perikarya in the SON; and (5) presence of activated AVP, OXY, and TH neurons in the periventricular, subependymal, and sub-PVN zones of the PVN. Topographic analysis revealed that the majority of AVP neurons expressing Fos occupied the dorsolateral and central part of the middle portion of the PVN. In the same PVN portion, Fos-OXY neurons occurred in similar frequency, however, they were primarily distributed along the lateral and medial margins of the PVN. In the SON, Fos-OXY cells occupied mainly its dorsal, while Fos-AVP cells predominated in its ventral part. The data clearly indicate that HS is not a selective stimulus neither for PVN nor SON itself and provide evidence that both PVN and SON AVP and OXY cells play important role in the mediation of signals induced by HS. In addition, the limited number of AVP, OXY, and TH neurons activated by HS may account for their differential functional specializations selective for stress/osmotic circuits activated by HS.     

Effect of hypertonic saline on the corticotropin-releasing hormone and arginine vasopressin content of the rat pituitary neurointermediate lobe

The changes in the levels of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) in the neurointermediate lobe of the pituitary (NIL) following hypertonic saline administration were examined in rats. The plasma osmotic pressure in rats receiving 2% NaCl for 8 days was greatly increased. Plasma AVP concentration in rats receiving 2% NaCl for 8 days were significantly higher than in control rats (566% of the control level). Plasma corticosterone was significantly higher in the saline-treated rats than in controls, whereas plasma ACTH was not significantly different. The pituitary ACTH concentration was much higher in the saline-treated rats than in controls. CRH in the NIL was increased significantly by saline treatment (419% of the control concentration), whereas the CRH in the paraventricular nucleus and median eminence of control and saline-treated rats did not differ significantly. The AVP in the NIL fell greatly in saline treated rats. The extract from both control and saline-treated rats showed a major peak for immunoreactive CRH, with a retention time identical to that of rat CRH. However, the peak was much higher in the extract from saline-treated rats. The immunoreactive AVP peak was greatly reduced in saline-treated rats. These results suggest that hypertonic saline administration increases the CRH in the NIL and causes AVP hypersecretion and/or hyperfunction of magnocellular-NIL CRH might be responsible for pituitary-adrenal stimulation in saline-treated rats.     

Effects of Morris water maze testing on the neuroendocrine stress response and intrahypothalamic release of vasopressin and oxytocin in the rat

Adult male Wistar rats were trained in the Morris water maze (MWM) on 3 consecutive days to find a visible platform. Concomitantly, microdialysis samples from the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei were collected in order to monitor local release of the neuropeptides vasopressin (AVP) and oxytocin (OXT), respectively, during controllable swim stress. Additionally, a separate set of animals was equipped with chronic jugular venous catheters to collect blood samples for analyzing plasma concentrations of corticotropin (ACTH) and corticosterone during training in the MWM. As measured by microdialysis, swimming in the MWM caused a significantly increased release of AVP within the PVN and of OXT within the SON on each of the 3 test sessions. In contrast to OXT in the SON, basal AVP concentrations in the PVN tended to rise from day to day. Plasma ACTH and corticosterone were found to be similarly elevated in response to MWM exposure on each of the test sessions. Taken together, these data demonstrate that testing in the MWM is not only associated with a significant activation of the hypothalamo-pituitary-adrenal axis but also with an intrahypothalamic release of AVP and OXT. If compared with findings using repeated forced swimming as an uncontrollable stressor (Wotjak, C.T., Ganster, J., Kohl, G., Holsboer, F., Landgraf, R., Engelmann, M., 1998. Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the secretory capacities of peptidergic neurons. Neuroscience 85, 1209-1222), the present results suggest that (1) similarities in the release profiles of AVP in the PVN and plasma hormone levels are fairly independent from the controllability of the stressor and seem, thus, to primarily relate to the physical demands of the task, whereas (2) the different intra-SON OXT release profiles might be linked to the controllability of the stressor.     

Vasopressin pressor receptor-mediated activation of HPA axis by acute ethanol stress in rats

The plasma arginine vasopressin (AVP), ACTH, and corticosterone levels and the hypothalamic corticotropin-releasing hormone (CRH) content were measured after oral administration of 1 ml of 75% ethanol to rats, a model known to induce acute gastric erosions and stress. Elevated plasma AVP, ACTH, and corticosterone levels were detected 1 h after ethanol administration. Treatment with the vasopressin pressor (V(1)) receptor antagonist [d(CH(2))(5)Tyr(Me)-AVP] before ethanol administration significantly reduced the ACTH and corticosterone level increases. A higher hypothalamic CRH content was measured at 30 or 60 min after ethanol administration. V(1) receptor antagonist injection, 5 min before ethanol administration, inhibited the rise in hypothalamic CRH content. The protein synthesis blocker cycloheximide prevented the hypothalamic CRH content elevation after stress. The AVP-, CRH-, and AVP + CRH-induced in vitro ACTH release in normal anterior pituitary tissue cultures was also prevented by pretreatment with the V(1) receptor antagonist. The results support the hypothesis that stress-induced AVP may not only act directly on the ACTH producing anterior pituitary cells but also indirectly at the hypothalamic level via the synthesis and release of CRH.     

Effect of vasopressin on Na+ kinetics in cultured rat vascular smooth muscle cells

The effect of arginine vasopressin (AVP) on Na+ kinetics was examined in cultured rat vascular smooth muscle cells (VSMC) and rat renal papillary collecting tubule cells (RPCT) by the direct measurement of intracellular sodium concentration [(Na+]i) using fluorescence dye; SBFI. AVP increased [Na+]i in a dose-dependent manner at a concentration of 10(-9) M or higher in rat VSMC but did not affect [Na+]i in rat RPCT. The calcium (Ca2+)-free solution completely blocked the increasing effect of AVP on [Na+]i in rat VSMC. A Ca2+ ionophore, ionomycin (1-2 x 10(-6) M) increased [Na+]i both in rat VSMC and RPCT. The Ca2(+)-free solution abolished the ionomycin-increased [Na+]i both in rat VSMC and RPCT. These results therefore indicate that after binding the V1 receptor AVP increases [Na+]i mediated through an increase in cellular Ca2+ uptake in VSMC.     

Effect of inhibition of Na+/K(+)-ATPase on the vasopressin-induced increase in intracellular Na+ concentration in vascular smooth muscle cells.

The effect of inhibition of Na+/K(+)-ATPase by ouabain on the arginine vasopressin (AVP)-induced increase in intracellular Na+ concentration [( Na+]i) was examined in cultured rat vascular smooth muscle cells (VSMC) by the direct measurement of [Na+]i using a fluorescent indicator dye. AVP at a concentration of 1 x 10(-9) M or higher increased [Na+]i in a dose-dependent manner in cultured rat VSMC. The preincubation of cells with 1 x 10(-4) M ouabain for 1 hr at 37 degrees C did not affect the basal [Na+]i but enhanced the 1 x 10(-6) M AVP-induced increase in [Na+]i. The preincubation was not necessary because similar results were obtained after the simultaneous administration of AVP and ouabain. The treatment with ouabain did not affect the intracellular pH changes induced by AVP. These results therefore indicate that the inhibition of Na+/K(+)-ATPase enhances the AVP-induced increase in [Na+]i by decreasing cellular Na+ efflux in cultured rat VSMC.     

Age-related changes in oxytocin-, arginine vasopressin- and nitric oxide synthase-expressing neurons in the supraoptic nucleus of the rat

Many histochemical investigations indicated that the oxytocin (OXY), the arginine vasopressin (AVP) and the nitric oxide synthase (NOS) have been synthesized in the supraoptic nucleus (SON) neurons. The objective of this study was to examine the age-related expression of the OXY, the AVP and the NOS in the SON of the young adult (2-month-old) and the aged (24-month-old) rats. The histochemistry for reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d; marker for the NOS) and the double labeling histochemistry for the OXY/NADPH-d or the AVP/NADPH-d were employed, and the quantitative analysis was performed with a computer-assisted image processing system. In comparison of the young adult and the aged group, the cell number, the cell size and the reactive density of the NOS-expressing neurons showed a significant increase along with age, and these evidences suggested the age-related increase of the nitric oxide (NO) production. The age-related significant increase was not detected in the number of the OXY/NOS-expressing neurons in the dorsal part, but was detected in the number of the AVP/NOS-expressing neurons in the ventral part. Based on our histochemical findings and reports demonstrated by other authors, we attempted to discuss the physiological role of NOS for the secretion of posterior pituitary hormones along with age.     

Oxytocin actions within the supraoptic and paraventricular nuclei: differential effects on peripheral and intranuclear vasopressin release

In response to forced swimming (FS), AVP is released somato-dendritically within the supraoptic nucleus (SON) and paraventricular nucleus (PVN), but not from neurohypophyseal terminals into blood. Together with AVP, oxytocin (OXT) is released within the SON and PVN. Here, we studied the role of intra-SON and intra-PVN OXT in the regulation of local AVP release and into the blood in male rats. Within the SON, bilateral retrodialysis of an OXT receptor antagonist (OXT-A) increased local AVP release in response to FS [60 s, 21 degrees C, vehicle twofold, not significant (ns); OXT-A: 15-fold increase, P < 0.05] without significantly affecting basal AVP release. In addition, local OXT-A elevated plasma AVP secretion under basal conditions (twofold increase, P < 0.05) without further elevation after FS. Within the PVN, exposure to FS elevated local AVP release, reaching significance only in the OXT-A group (vehicle: 1.4-fold, ns; OXT-A: 1.6-fold increase, P = 0.050). Bilateral OXT-A into the PVN did not affect peripheral AVP secretion either under basal or stress conditions. Basal ACTH concentrations tended to be elevated by local OXT-A within the PVN (1.7-fold increase, P = 0.076). In contrast, the swim-induced ACTH secretion was attenuated after retrodialysis of OXT-A within both the SON (at 5 min) and PVN (at 15 min) (P < 0.05 both) compared with vehicle. The results demonstrate a receptor-mediated effect of OXT within the SON and PVN on local and neurohypophyseal AVP release, which depends upon the activity conditions. Further, while exerting an inhibitory effect on hypothalamo-pituitary-adrenal axis activity under basal conditions, hypothalamic OXT is essential for an adequate acute ACTH response.     

Elevated [Cl-]i, and [Na+]i inhibit Na+, K+, Cl- cotransport by different mechanisms in squid giant axons

Bumetanide-sensitive (BS) unidirectional fluxes of (36)Cl- or (22)Na+ were measured in internally dialyzed squid giant axons while varying the intra- or extracellular concentrations of Na+ and/or Cl-. Raising either [Cl-]i or [Na+]i resulted in a concentration-dependent reduction of the BS influx of both (36)Cl- and (22)Na+. Raising [Cl-]i above 200 mM completely blocked BS influxes. However, raising [Na+]i to 290 mM resulted in saturable but incomplete inhibition of both BS Na+ influx and BS Cl- influx. The consequences of varying intracellular Cl- on cotransporter effluxes were complex. At lower [Cl-]i values (below 100 mM) intracellular Cl- activated cotransporter effluxes. Surprisingly, however, raising [Cl-]i levels > 125 mM resulted in a [Cl-]i-dependent inhibition of BS effluxes of both Na+ and Cl-. On the other hand, raising [Na+]i resulted only in the activation of the BS Na+ efflux; intracellular Na+ did not inhibit BS efflux even at 290 mM. The inhibitory effects of intracellular Na+ on cotransporter-mediated influxes, and lack of inhibitory effects on BS effluxes, are consistent with the trans-side inhibition expected for an ordered binding/release model of cotransporter operation. However, the inhibitory effects of intracellular Cl- on both influxes and effluxes are not explained by such a model. These data suggest that Cl may interact with an intracellular site (or sites), which does not mediate Cl transport, but does modulate the transport activity of the Na+, K+, Cl- cotransporter.     

The cellular actions of vasopressin on corticotrophs of the anterior pituitary: resistance to glucocorticoid action

The cellular actions of vasopressin (AVP) in the anterior pituitary were investigated. HPLC analysis of [3H]inositol-labeled cells indicated that AVP stimulated a rapid increase in inositol-1,4,5 trisphosphate (IP3), inositol-1,4 bisphosphate, and inositol-4 monophosphate levels. While CRF had no effect on basal IP3 levels, it blocked their stimulation by AVP. CRF-stimulated ACTH secretion and cAMP accumulation were potentiated by AVP. AFter dexamethasone (DEX) treatment (20 nM, 18 h), CRF-dependent ACTH secretion and cAMP accumulation were attenuated but AVP was still able to potentiate both of these actions of CRF suggesting that cellular actions of AVP may be resistant to DEX effects. Therefore, [3H]AVP binding was determined in control and DEX-treated cells. Pretreatment with DEX had no effect on either AVP receptor affinity or on the number of available binding sites. Consistently, stimulation of IP3 production by AVP in DEX-treated cells was comparable to that of control cells. Protein kinase C activators such as 12-O-tetradecanoyl-phorbol-13-acetate and dioctanoylglycerol were either near additive with CRF or also potentiated the action of CRF on ACTH secretion, respectively, even after DEX pretreatment. These results indicate that, in the anterior pituitary, distinct intracellular signaling pathways mediate the actions of CRF and AVP; cAMP mediates CRF actions and IP3/protein kinase C mediate the effects of AVP. Neuromodulation of ACTH secretion by dual effector mechanisms which exhibit a complex mode of interaction and only one of which is negatively influenced by glucocorticoids, provides these cells a mechanisms by which appropriate responses can be elicited under various physiological states.     

Delineation of responsive AVP-containing neurons to running stress in the hypothalamus

Running becomes a stress, termed running stress, if it persists above the lactate threshold (LT) and results in enhanced plasma ACTH level in humans. Although the exact underlying regulation mechanism is still uncertain, hypothalamic AVP has been shown to play a dominant role in running-induced ACTH release. It is still not known, however, whether running stress activates the hypothalamic AVP-containing neurons that are involved in the activation of the ACTH response. For this reason, we applied our rat running stress model, in which both plasma ACTH and osmolality levels increase just above LT running (supra-LT running), to delineate which hypothalamic AVP neurons were responsive to running stress. Rats were previously habituated to running and then subjected to a 30-min run either just below or above the LT. Plasma samples were collected from these animals to determine ACTH and osmolality levels. Brains were prepared for immunocytochemistry for both AVP/Fos in the hypothalamus and enzyme immunoassay for the stalk median eminence (SME) AVP content. Only supra-LT running resulted in an increase in the number of Fos/AVP-immunoreactive neurons in both the parvocellular paraventricular nucleus (pPVN) and the magnocellular supraoptic nucleus (SON) accompanied by increased ACTH and plasma osmolality levels. Similarly, running reduced the SME content of the AVP. We thus found that AVP-containing neurons located in both the pPVN and SON are responsive to running stress just above the LT.     

Stress mediated changes in hypothalamic corticotropin releasing factor-like immunoreactivity

Hypothalamic corticotropin releasing factor-like immunoreactivity (CRF-LI), plasma ACTH and corticosterone levels were measured by radioimmunoassay over a two hour period of restraint stress. The results of this study demonstrate a significant decrease in hypothalamic CRF-LI levels 15 and 30 minutes after the start of restraint stress which is followed by a significant increase at 60 minutes that is abolished by cycloheximide pretreatment. Plasma ACTH and corticosterone levels were significantly elevated after 15, 30, 60, 90, and 120 minutes of restraint stress. These results are consistent with a release of CRF from the hypothalamus during stress. The cycloheximide-sensitive increase in hypothalamic CRF-LI indicates that synthesis of CRF-41 occurs during prolonged stress. These results suggest that the response of an organism to exposure to a long-term, high intensity stress involves both the release and synthesis of CRF-41.     

A comparison of grooming behavior potencies of neurohypophyseal nonapeptides

We have previously demonstrated that intracerebroventricular (ICV) administration of oxytocin (OXY) enhanced grooming behaviors in male and female rats at a 1 microgram dose. In the present study female rats were injected ICV with 1 microgram OXY or equimolar doses of other peptides. At this dose arginine-vasopressin (AVP), arginine-vasotocin (AVT) and lysine-vasopressin (LVP), as well as alpha-MSH, were as effective as OXY in increasing grooming behavior. At equimolar doses, ACTH1-10, tocinoic acid (the ring structure of OXY) and Pro-Leu-Gly-NH2 (the tail structure of OXY) had no significant effect on grooming behavior. The potency of AVP and AVT was determined across a 0.05-5 microgram dose range. Grooming scores increased in an apparent linear manner across a similar OXY dose range. Both AVP and AVT, however, manifested an inverted U grooming response curve. Maximum grooming scores resulted from a 0.1 microgram dose of AVT or a 0.5 microgram AVP dose. Analyses of the aspects of grooming separately found that nonapeptides OXY, AVP and AVT all elevated body grooming, washing, and scratching. Because AVT and AVP administration resulted in grooming scores significantly higher than OXY at lower doses, we concluded that the CNS is more sensitive to the effects of AVT and AVP on grooming behavior than OXY.     

Permeability properties and intracellular ion concentrations of epithelial cells in rat duodenum.

Effects of the K+ concentration in the bathing fluid ([K+]l) on the intracellular K+, Na+ and Cl- concentrations ([K+]i [Na+]i and [Cl-]i) as well as on the electrical potential were studied in rat duodenum. Changes in the mucosal K+ concentration ([K+]m), bringing the sum of Na+ and K+ concentrations to 147.2 mM constant, had little effect on the transmural potential difference (PDt), but did induce marked changes in the mucosal membrane potential (Vm). As [K+]m increased, Vm was depolarized gradually and obeyed the Nernst equation for a potassium electrode in the range of [K+]m greater than approx. 60 mM. Experiments of ion analyses were carried out on strips of duodenum to determine the effect of changing the external K+ concentrations on [K+] i, [Na+]i and [Cl-]i. An increase in [K+]o resulted in increases in [K+]i and [Cl-]i and a decrease in [Na+]i, [K+]i approaching its maximum at [K+]o greater than 70 mM. Such changes in [K+]i and [Na+]i seem to correlate quantitatively with the changes in [K+]o and [Na+]o. The values of the ratio of permeability coefficients, Pna+/PK+ were estimated using the Vm values and intracellular ion concentrations measured in these experiments. The results suggested that there appeared a rather abrupt increase in the PNa+/PK+ ratio from 0 to approx. 0.1, as [K+]m decreased.     

Comparison of changes evoked by GABA (gamma-aminobutyric acid) and anoxia in [K+]o, [Cl-]o, and [Na+]o in stratum pyramidale and stratum radiatum of the guinea pig hippocampus

Ion-selective microelectrode recordings were made to assess a possible contribution of extracellular gamma-aminobutyric acid (GABA) accumulation to early responses evoked in the brain by anoxia and ischemia. Changes evoked by GABA or N2 in [K+]o, [Cl-]o, [Na+]o, and [TMA+]o were recorded in the cell body and dendritic regions of the stratum pyramidale (SP) and stratum radiatum (SR), respectively, of pyramidal neurons in CA1 of guinea pig hippocampal slices. Bath application of GABA (1-10 mM) for approximately 5 min evoked changes in [K+]o and [Cl-]o with respective EC50 levels of 3.8 and 4.1 mM in SP, and 4.7 and 5.6 mM in SR. In SP 5 mM GABA reversibly increased [K+]o and [Cl-]o and decreased [Na+]o; replacement of 95% O2 -5% CO2 by 95% N2 -5% CO2 for a similar period of time evoked changes which were for each ion in the same direction as those with GABA. In SR both GABA and N2 caused increases in [K+]o and decreases in [Cl-]o and [Na+]. The reduction of extracellular space, estimated from levels of [TMA+]o during exposures to GABA and N2, was 5-6% and insufficient to cause the observed changes in ion concentration. Ion changes induced by GABA and N2 were reversibly attenuated by the GABA(A) receptor antagonist bicuculline methiodide (BMI, 100 microM). GABA-evoked changes in [K+]o in SP and SR and [Cl-]o in SP were depressed by > or =90%, and of [Cl-]o in SR by 50%; N2-evoked changes in [K+]o in SP and SR were decreased by 70% and those of [Cl-]o by 50%. BMI blocked delta [Na+]o with both GABA and N2 by 20-30%. It is concluded that during early anoxia: (i) accumulation of GABA and activation of GABA(A) receptors may contribute to the ion changes and play a significant role, and (ii) responses in the dendritic (SR) regions are greater than and (or) differ from those in the somal (SP) layers. A large component of the [K+]o increase may involve a GABA-evoked Ca2+-activated gk, secondary to [Ca2+]i increase. A major part of [Cl-]o changes may arise from GABA-induced g(Cl) and glial efflux, with strong stimulation of active outward transport and anion exchange at SP, and inward Na+/K+/2Cl- co-transport at SR. Na+ influx is attributable mainly to Na+-dependent transmitter uptake, with only a small amount related to GABA(A) receptor activation. Although the release and (or) accumulation of GABA during anoxia might be viewed as potentially protectant, the ultimate role may more likely be an important contribution to toxicity and delayed neuronal death.     

Roles of CO2, O2, and acid in arteriovenous [H+] difference during muscle contractions

To determine the origins of the arteriovenous [H+] difference of muscle during contractions, arterial and muscle venous blood sample pairs were taken before and after 0.5, 5.0, and 30.0 min of 4/s isometric twitches of the gastrocnemius-plantaris muscle group of anesthetized dogs. These samples were analyzed for PO2, PCO2, and pH, the concentrations of O2, CO2, K+, Na+, La-, and Cl- in whole blood, and La-, K+, Na+, and Cl- in plasma. Whole blood was hemolyzed and analyzed for PO2, PCO2, and pH. Net O2 uptake, CO2 output, L, K+, Na+, and Cl- were calculated in addition to net output of non-CO2 acid (HA) and strong ion difference ([SID]) and common ion [SID] ([K+] + [Na+] - [Cl-] - [La-]). From these data we partitioned the origins of the arteriovenous [H+] difference via the common PCO2-pH diagram and via a [H+]-PCO2 diagram and determined whether true plasma arteriovenous [H+] differences reflect plasma and cell arteriovenous [H+] differences. The arteriovenous [H+] differences of plasma and hemolyzed blood were the same, showing that true plasma does reflect plasma and cells. K+ showed a small significant but transient output. Na+ was not significant, whereas Cl- showed a significant transient uptake. Lactate output and HA, calculated for dog blood acid-base, showed transient outputs and were the same. At 5.0 min when the arteriovenous difference was largest, CO2 alone would have increased [H+] 15.9 nmol/l whereas desaturation of Hb would have decreased [H+] 4.2 nmol/l and lactate could have raised [H+] 1.0 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)