Autoregulation of follicle-stimulating hormone secretion in the hamster: evidence for control at the level of the anterior pituitary gland.

Pituitary tissue obtained from proestrous, phenobarbital-treated hamsters was placed in organ culture, and the release rates of FSH and LH were monitored. Addition of LHRH to the culture medium increased gonadotropin release rates. Preincubation of pituitaries in medium that contained 1 microgram purified FSH ( NIADDK rat FSH-I-5) resulted in enhanced basal and LHRH-induced FSH release rates. Full expression of FSH hypersecretion by pituitary tissue occurred after 3-h exposure to purified FSH. This phenomenon appears to require adequate tissue calcium concentrations. Further, exposure of pituitaries to purified FSH slightly but significantly enhanced basal but not LHRH-stimulated LH release compared with that in untreated controls. Experiments that employed labeled FSH, reduced exposure time to purified FSH, or decreased calcium concentration in the medium proved that the increased FSH secretory rate was not due to contamination of the medium with the purified FSH used to stimulate the pituitary. These studies suggest that FSH has the ability to influence its own secretion by an action at the level of the anterior pituitary gland.

Constitutive expression of interleukin 4 in vivo does not lead to the development of T helper 2 type CD8+ T cells secreting interleukin 4 or interleukin 5.

Interleukin 4 (IL-4) has been shown to commit CD8+ T cells to a T helper (Th) 2 functional phenotype in vitro. To study the effects of IL-4 on CD8+ T cell development in vivo we analysed the CD8+ T cell phenotype in mice constitutively expressing IL-4. Purified CD8+ T cells from uninfected or flu infected IL-4 transgenic (tg) animals produced no detectable IL-4 or IL-5 after in vitro stimulation on anti-CD3 coated plates. However, CD8+ T cells from IL-4 tg mice could be converted into IL-4 and IL-5 producers in vitro in the presence of exogenous added IL-4, showing that these cells were still responsive to IL-4. IL-4 tg mice also showed a delay in influenza virus clearance from the lung, which was probably due to the observed reduction of total CD8+ T cell numbers in the IL-4 tg animals since IL-4 tg CD8+ T cells showed normal levels of influenza-specific cytotoxicity in comparison to controls. Taken together these results suggest that CD8+ T cells are not necessarily switched to a Th2 phenotype by the presence of IL-4 and that some other factor(s) may be important in the switch process of CD8+ T cells in vivo, since the addition of IL-4 during CD8+ T cell activation in vitro leads to Th2 type CD8+ T cells secreting IL-4 and IL-5.

No evidence for differences between pre- and post-junctional alpha 2-adrenoceptors.

We have examined the pre- and post-junctional effects of a series of alpha-adrenoceptor agonists and antagonists at alpha 2-adrenoceptors in the pithed rat preparation and the human isolated saphenous vein. In the pithed rat, there was no difference in relative agonist and antagonist potencies between pre- and post-junctional alpha 2-adrenoceptors but the absolute potencies of antagonists differed: antagonists were more potent prejunctionally. In the human saphenous vein, the alpha 2-adrenoceptor antagonist yohimbine had pre- and post-junctional actions over the same concentration range. We have no evidence to suggest differences between pre- and post-junctional alpha 2-adrenoceptors: differences in absolute antagonist potencies in the pithed rat may be due to non-equilibrium conditions.

Evidence for neuro-effector transmission through postjunctional alpha 2-adrenoceptors in human saphenous vein.

The effects of the alpha 1-adrenoceptor antagonist prazosin and the alpha 2-adrenoceptor antagonist yohimbine were examined against stimulation-evoked contractions in human isolated saphenous veins. The concentration of yohimbine producing 30% inhibition of stimulation-evoked contractions (IC30) was 13.2 nM, whereas the IC30 of prazosin was greater than 250 nM. The inhibition of stimulation-evoked contractions by yohimbine was not prejunctionally mediated since yohimbine (0.01-0.1 microM) significantly potentiated the stimulation-evoked overflow of tritium in tissues pre-incubated with [3H]-noradrenaline. The high potency of yohimbine and the low potency of prazosin indicate that neuro-effector transmission in human saphenous vein is mediated predominantly by postjunctional alpha 2-adrenoceptors.

An examination of 5-hydroxytryptamine receptors in human saphenous vein.

We have examined the effects of antagonists on the isometric contraction of the human saphenous vein produced by 5-hydroxytryptamine (5-HT). The 5-HT2-antagonist ketanserin (1 microM) had little effect on the lower part of the concentration-response curve to 5-HT, but markedly shifted the upper part of the curve. Yohimbine caused an approximately parallel shift of the concentration-response curve to 5-HT, with a pA2 of 5.48, much lower than its pA2 against noradrenaline in the absence (6.36) or presence (7.06) of cocaine. It is concluded that there are two components to the contractile response to 5-HT in human saphenous vein: at low concentrations 5-HT activates a yohimbine-sensitive receptor, and at higher concentrations 5-HT activates a 5-HT2-receptor.

Noninvasive flow techniques in the diagnosis of cerebrovascular disease.

The accuracy of oculoplethysmography (OPG) and carotid phonoangiography (CPA) singly and in combination, the Doppler velocity detector, and photoplethysmography (PPG) was checked by measurement of the degree of stenosis as shown on arteriograms in 308 internal carotid arteries. In a second study using arteriographic measurement in 210 internal carotid arteries, the comparative accuracy of the fluid-filled (Kartchner) and the air-filled (Zira) OPG, each with and without CPA, was assessed. In the first study the specificity in arteries with less than 40% diameter reduction varied from 88% for the PPG to 97% for the Doppler examination. The sensitivity in arteries with more than 40% diameter reduction varied from 17% for the Doppler examination to 80% for the combination of OPG plus CPA. For arteries with a reduction in diameter greater than 70%, the sensitivity varied from 67% for the CPA to 87% for the OPG plus CPA. The sensitivity of the OPG plus CPA for total occlusions was 93%. For bilateral carotid artery stenosis over 40%, the sensitivity varied from 50% for the CPA to 82% for the combined OPG plus CPA. In the second study, for arteries with less than 40% stenosis the specificity varied from 86% for the Zira computed readout to 93% for the OPG(K). In the second study, when retrospectively analyzed, the sensitivity for arteries with more than 40% stenosis varied from 74% for the Zira computed readout to 88% for the combined OPG(K) plus CPA. For arteries with greater than 70% diameter reduction the sensitivity varied from 79% for the Zira readout to 100% for OPG plus CPA. For bilateral carotid artery disease with greater than 40% diameter reduction, the sensitivity ranged from 50% for OPG(Z) to 77% for OPG(Z) plus CPA.

The axillo-axillary bypass graft: further experience.

The recent utilization of the axillo-axillary bypass graft for revascularization of proximal subclavian or innominate artery occlusions has proved to be a successful operation, technically simple with a low operative morbidity. We have reviewed the reported experience to date and have added our experience of nine additional cases. The procedure has produced excellent results in all patients, with no evidence of postoperative "steal" from the donor limb. Preoperative hemodynamic defects were corrected. Our experience has led us to the conclusion that this simpler and safer extrathoracic approach should be considered as the procedure of choice when arterial lesions allow for its selection and use.

Alpha-adrenoceptor responsiveness in the aged rat.

The effects of ageing on vascular alpha 1- and alpha 2-adrenoceptors were examined using anaesthetised and pithed young (3-7 months) and old (21-24 months) Sprague-Dawley rats. In pithed animals, the pressor and cardioinhibitory effects of the alpha 2-adrenoceptor agonist xylazine were significantly reduced in old animals (8- and 6-fold shift), but the pressor effects of the alpha 1-adrenoceptor agonist amidephrine were not significantly altered by ageing. In anaesthetised rats, the pressor response to the mixed alpha-adrenoceptor agonist noradrenaline (NA) was not significantly altered, and the pressor potency of the alpha 1-adrenoceptor agonist amidephrine was only slightly reduced (1.7-fold shift) in old animals. In the presence of cocaine (1 mg kg-1) the pressor potency of NA was markedly reduced (13-fold shift) in old animals. In the presence of prazosin (1 mg kg-1) to eliminate alpha 1-adrenoceptor-mediated responses, the pressor potency of NA was markedly reduced in old as animals as compared to young animals (16-fold shift). The neuronal uptake blocker cocaine (1 mg kg-1) significantly potentiated the pressor response to NA only in young. In summary, we have found a reduced alpha 2-adrenoceptor-mediated pressor and cardioinhibitory responsiveness and a reduced neuronal uptake of NA in old animals with little change in alpha 1-adrenoceptor-mediated vascular responsiveness.

Further examination of the effects of ageing on the adrenoceptor responsiveness of the rat vas deferens.

Age-related changes in presynaptic alpha 2-adrenoceptors were investigated in prostatic and epididymal portions of vasa deferentia from young adult (2-3 months) and old (24-29 months) Sprague-Dawley rats, using the alpha 2-selective agonists xylazine and clonidine. In prostatic portions the inhibitory effects of clonidine against the isometric contractions to single pulse field stimulation were complicated by a postsynaptic action in old animals, but in epididymal portions in the presence of nifedipine both xylazine and clonidine were 3 times less potent in old rats. However, there were no significant differences between young and old in the potency of xylazine at inhibiting the overflow of tritium or the isometric contraction evoked by 5 Hz stimulation for 3 min in tissues pre-incubated with [3H]noradrenaline. It is suggested that there is reduced responsiveness of presynaptic alpha 2-adrenoceptors in the vas deferens of old rats, but that this can only be demonstrated using a sensitive measure of the presynaptic potency of agonists: the isometric contraction to a single stimulus.

An investigation of age-related changes in pre- and postjunctional alpha-adrenoceptors in human saphenous vein.

The responsiveness of prejunctional alpha 2-, postjunctional alpha 1- and postjunctional alpha 2-adrenoceptors was examined in human isolated saphenous veins from male patients in the age range 37-70. There was no age-related alteration in the prejunctional potency of the alpha 2-adrenoceptor agonist xylazine for inhibiting the stimulation-evoked overflow of tritium in tissues preincubated with [3H]noradrenaline. The alpha 2-adrenoceptor antagonist yohimbine (0.01-1 microM) and the alpha 1-adrenoceptor antagonist prazosin (0.1-1 microM) significantly reduced stimulation-evoked contractions in a concentration-dependent manner. There was no significant age-related correlation for the potency of prazosin but there was a significant negative correlation between the potency of yohimbine and age (r = 0.70, n = 11, P less than 0.05), i.e. the potency of yohimbine decreased with increasing age. The decreased postjunctional potency of yohimbine may reflect a loss of alpha 2-adrenoceptors with increasing age.

An investigation of the actions of the calcium entry facilitator Bay K 8644 on the rat vas deferens.

The pre- and postsynaptic actions of the calcium entry facilitator Bay K 8644 were investigated in the rat isolated vas deferens. Bay K 8644 had no presynaptic effect on adrenergic neurotransmission in the epididymal portion. Bay K 8644 and alpha 1-adrenoceptor agonists potentiated, and the calcium entry blocker nifedipine abolished, the non-adrenergic contraction to a single stimulus in prostatic portions. Bay K 8644 sensitised the prostatic portion to, but did not increase the maximum height of, spontaneous direct contractions produced by alpha-agonists.

Further examination of the inhibitory actions of alpha 1-adrenoceptor agonists in rat vas deferens.

The inhibitory actions of alpha 1-adrenoceptor agonists were examined in the isolated bisected vas deferens of the rat. The calcium entry facilitator Bay K 8644 markedly potentiated the isometric contraction to a single stimulus pulse in epididymal portions of rat vas deferens: subsequent amidephrine produced an inhibition which was antagonised by the alpha 1-adrenoceptor antagonist, prazosin, but not by the alpha 2-adrenoceptor antagonist, yohimbine. The alpha 1-adrenoceptor agonists amidephrine and cirazoline failed to inhibit the transmitter overflow to trains of pulses at a frequency of 2 Hz in epididymal portions, but also failed to abolish the nifedipine-resistant adrenergic contraction to trains of pulses at 2 Hz in epididymal portions. It is concluded that alpha 1-adrenoceptor agonists have inhibitory effects which may be by action at presynaptic alpha 1-adrenoceptors.

Pituitary follicle-stimulating hormone heterogeneity: assessment of biologic activities of each follicle-stimulating hormone form.

The multiple species of follicle-stimulating hormone (FSH) present within pituitary tissue were separated by the technique of polyacrylamide gel isoelectric focusing. The ability of each FSH species to stimulate the secretion of plasminogen activator from cultured granulosa cells was tested (FSH in vitro bioassay). A wide range of biologic/radioimmunologic FSH activity was observed when FSH species were compared. As the isoelectric point of the FSH molecule declined, so did the biologic activity. A second series of studies was performed to determine which forms of FSH were secreted by pituitary tissue in vitro. All of the forms of FSH present in pituitary tissue were secreted into culture medium. However, the relative proportions of FSH forms in the pituitary and medium were not always similar. Exposure of pituitary tissue to luteinizing hormone-releasing hormone elicited an increase in the relative proportion of the more biologically active forms of FSH that were secreted. These studies suggest that the hormonal milieu surrounding the pituitary affects not only the quantity but also the potency of the FSH signal emitted. Thus, the basis for observed differences between biologic and immunologic FSH activities observed during some endocrine states may be the result of preferential secretion of certain FSH species.

Age-related alterations in alpha 1- and beta-adrenoceptors mediated responsiveness of rat aorta.

We have examined the responsiveness of alpha- and beta-adrenoceptors in aortae from 1.5, 3, 6 and 24 month old rats. The isometric contraction to phenylephrine was antagonised competitively by prazosin with a pA2 value of 9.45, suggesting that the receptor is an alpha 1-adrenoceptor. The potency of phenylephrine was significantly reduced in 24 months old as compared with all younger rats combined. The maximum contraction to phenylephrine was unaltered in 24 month old rats. The maximum contraction to potassium chloride was significantly less than that to phenylephrine only in 1.5 months old rats. In tissues contracted by potassium chloride, isoprenaline produced a marked relaxation in 1.5 months old animals, but there was a progressive loss with increasing age of the beta-adrenoceptor-mediated relaxation which was markedly reduced by 6 months and abolished in 24 months old. It is concluded that, in the rat aorta, there is a decrease in alpha 1-adrenoceptor responsiveness in senescence, and a loss of beta-adrenoceptor-mediated responses in maturation.

Adaptation of the plasma renin radioimmunoassay for use with HIV-1 protease.

We have demonstrated the use of a radioimmunoassay to quantitate the peptidolytic activity of human immunodeficiency virus, type 1 (HIV-1) protease using a tetradecapeptide substrate of porcine renin, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser. HIV-1 protease catalyzes cleavage of this substrate at the same Leu-Leu bond as does porcine renin, resulting in the formation of authentic angiotensin-I. The angiotensin-I product is then detected by use of a commercially available renin plasma assay kit, which constitutes the basis of the RIA. The radioimmunoassay provides detection of the protease-catalyzed formation of angiotensin-I at picomolar concentrations in vitro. We demonstrate the use of this assay in determining IC50 values for two HIV-1 protease inhibitors present in cell culture media and in standard assay buffer. An example of the potential development of this assay for the quantitation of these inhibitors present in ex vivo plasma samples is also presented.

Nasal-associated lymphoid tissue is a site of long-term virus-specific antibody production following respiratory virus infection of mice.

Nasal immunoglobulin A provides an initial defense against inhaled respiratory pathogens. However, it is not known whether the nasal-associated lymphoid tissues (NALT) are able to mount an effective long-lasting pathogen-specific immune response, nor is it known whether functional differences exist between the organized NALT (O-NALT) and the diffuse NALT lining the nasal passages (D-NALT). Here we show that although both the O-NALT and the D-NALT are capable of producing virus-specific antibody in response to influenza virus infection, the frequency of specific antibody-forming cells in the D-NALT is much greater than the frequency observed in the O-NALT. Furthermore, we show that the D-NALT but not the O-NALT is the site of long-term virus-specific humoral immunity which lasts for the life of the animal. These results indicate that the D-NALT is not only the major effector site of the NALT but also the site of local long-term specific antibody production.

Vascular alpha 2-adrenoceptors can mediate nerve stimulation-evoked contractions.

It is now established that alpha 2-adrenoceptors can be present on vascular smooth muscle as targets for exogenous agonists, but some controversy exists as to whether these vascular alpha 2-receptors may also be the target for neurally released noradrenaline. In human saphenous vein we have found evidence that the alpha 2-antagonist yohimbine in low concentrations (0.01 mumol/l) can significantly inhibit stimulation-evoked contractions, whereas the alpha 1-antagonist prazosin is more than ten times less potent. It is concluded that, at least in some tissues, neuro-effector transmission can be mediated by vascular alpha 2-adrenoceptors.

Regulation of the pulsatile releases of luteinizing and follicle-stimulating hormones in ovariectomized hamsters.

We have combined for modifications of common radioimmunoassay (RIA) techniques to increase the sensitivity of the gonadotropin assays by an order of magnitude compared with those generated according to the instructions provided by the National Pituitary Agency. The four modifications are: a) enzymatic radioiodination, b) purification of radiolabeled hormones by Sephadex and concanavalin A chromatography, c) reduced first antibody concentration, and d) a prolonged incubation time. These methods increase the sensitivities of the RIAs and allow for the quantitation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels in small volumes of plasma. We have used these methods to measure the changes in pulse frequency and amplitude of LH and FSH in ovariectomized hamsters after a variety of neuroendocrine manipulations. Alterations in catecholaminergic neurotransmission affect the frequency and amplitude of LH but not FSH release, and suggest that the hypothalamic mechanisms responsible for LH releasing hormone (LHRH)-mediated LH release are distinct from those that regulate FSH secretion. Further, alterations in LHRH-pituitary interactions (elicited by injections of LHRH antisera or a potent LHRH agonist), suggest the existence of separate control mechanisms responsible for LH and FSH release at the level of the adenohypophysis. Combined, these studies provide further evidence for complex and separate neuroendocrine regulatory control over the secretion of each gonadotropin.

Human immunodeficiency virus-1 protease. 2. Use of pH rate studies and solvent kinetic isotope effects to elucidate details of chemical mechanism.

The pH dependence of the peptidolytic reaction of recombinant human immunodeficiency virus type 1 protease has been examined over a pH range of 3-7 for four oligopeptide substrates and two competitive inhibitors. The pK values obtained from the pKis vs pH profiles for the unprotonated and protonated active-site aspartyl groups, Asp-25 and Asp-25', in the monoprotonated enzyme form were 3.1 and 5.2, respectively. Profiles of log V/K vs pH for all four substrates were "bell-shaped" in which the pK values for the unprotonated and protonated aspartyl residues were 3.4-3.7 and 5.5-6.5, respectively. Profiles of log V vs pH for these substrates were "wave-shaped" in which V was shifted to a constant lower value upon protonation of a residue of pK = 4.2-5.2. These results indicate that substrates bind only to a form of HIV-1 protease in which one of the two catalytic aspartyl residues is protonated. Solvent kinetic isotope effects were measured over a pH (D) range of 3-7 for two oligopeptide substrates, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 and Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2. The pH-independent value for DV/K was 1.0 for both substrates, and DV = 1.5-1.7 and 2.2-3.2 at low and high pH (D), respectively. The attentuation of both V and DV at low pH (D) is consistent with a change in rate-limiting step from a chemical one at high pH (D) to one in which a product release step or an enzyme isomerization step becomes partly rate-limiting at low pH (D). Proton inventory data is in accord with the concerted transfer of two protons in the transition state of a rate-limiting chemical step in which the enzyme-bound amide hydrate adduct collapses to form the carboxylic acid and amine products.