Adjunctive treatment of partial seizures with tiagabine: a placebo-controlled trial.

Tiagabine is a new antiepileptic drug which acts by a novel mechanism, inhibiting the reuptake of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) into neurons and glia. A double-blind, placebo-controlled, crossover trial was undertaken, based upon a response-dependent design. Ninety-four patients with complex partial seizures with or without secondary generalised tonic-clonic seizures were recruited into an open screening phase and tiagabine was added to their existing drug therapy in doses titrated to reduce seizure frequency by > or = 25% or to the limit of tolerance. Forty-six responders were subsequently randomised to a double-blind crossover trial in which tiagabine was compared with placebo. Forty-two patients completed the trial. A significant reduction in the frequency of complex partial and secondary generalised tonic clonic seizures was seen. Twenty-six percent had a reduction of > or = 50% in the frequency of their complex partial seizures, and of the 27 patients who also had secondary generalised tonic clonic seizures, 63% experienced a reduction of > or = 50%. No interactions with baseline antiepileptic drugs were detected and no serious adverse reactions occurred. The commonest adverse events were tiredness, dizziness and headache. We conclude that tiagabine has promising antiepileptic effects. Further trials are underway.

Involvement of a metalloprotease in low-affinity nerve growth factor receptor truncation: inhibition of truncation in vitro and in vivo.

The mechanism of low-affinity NGF receptor (LNGFR) truncation was investigated in cultured Schwann cells. Affinity labeling of Schwann cells with 125I-NGF or metabolic labeling with 35S-cysteine showed that truncated NGF receptor (NGF-Rt) was derived from the cell surface form of the receptor. Addition of full-length, exogenous NGF receptor (M(r) = 80 kDa) to Schwann cell membranes resulted in cleavage of the exogenous substrate to NGF-Rt. Investigations into the mechanism of truncation revealed that metalloprotease inhibitors such as phenanthroline, bathophenanthroline, and 8-hydroxyquinoline (8-OHQ) blocked LNGFR truncation in a concentration-dependent fashion. Inhibitors of other protease classes had no effect on truncation. In addition, truncation did not occur at 4 degrees C. It was found that truncation could also occur in Schwann cell membrane preparations, indicating that the putative protease was membrane bound and closely associated with the LNGFR. Metal reconstitution experiments revealed a strong preference toward zinc for the truncating activity, with iron and manganese having slight reconstitution activity in phenanthroline-quenched membranes. To determine if apparent truncation could be inhibited in vivo, the metalloprotease inhibitor 8-OHQ was administered to neonatal rats. 8-OHQ resulted in decreased urine and blood NGF-Rt levels and increased the sciatic nerve LNGFR content; this effect was dose dependent. In adult rats with sciatic nerve crush lesions, 8-OHQ (30-300 mg/kg, t.i.d.) significantly enhanced the rate of sensory neuron regeneration as assessed by the nerve pinch assay. This was accompanied by increased levels of LNGFR in distal nerve segments. These results suggest that Schwann cells possess a metalloprotease-like activity that serves to cleave LNGFR from the surface of these cells. We propose that the putative metalloprotease represents a novel mechanism by which the Schwann cell regulates this particular cell surface protein. Furthermore, increasing the amount of Schwann cell surface LNGFR appears to be of functional significance in that sensory nerve regeneration can be enhanced by inhibition of truncation.

Actions of A-75200, a novel catecholamine uptake inhibitor, on norepinephrine uptake and release from bovine adrenal chromaffin cells.

The balance between catecholamine (CA) release and reuptake is closely regulated and determines the effective level of transmitter at the synaptic cleft. Drugs that block CA uptake have potential utility as antidepressant medications. One such drug is racemic (+/-)-(1' R*,3R*)-3-phenyl-1-[1',2',3',4'-tetrahydro-5',6'- methylenedioxy-1'-naphthalenyl-methyl]-pyrrolidine methanesulfonate (A-7500), a novel polycyclic compound developed at Abbott Laboratories. This compound is known to bind to CA transporters in the central nervous system, however, its effects on an intact neurosecretory system have not been studied. In this regard, norepinephrine (NE) release from bovine adrenal chromaffin cells (BACC) is a classic model system for CA release and is an excellent system in which to examine the effects of drugs which modulate neurotransmitter release. We compared the effects of A-75200 and its two constituent enantiomers, A-74111 and A-74112, to the effects of three well-characterized uptake inhibitors, desipramine (DMI), nomifensine and cocaine. We found that the Abbott compounds inhibit [3H]norepinephrine ([3H]NE) uptake with an EC50 comparable to cocaine. In addition, unlike nomifensine and cocaine, these compounds inhibited nicotine- and K(+)-stimulated NE release, whereas histamine-stimulated release was preserved. Thus, the Abbott compounds block the effects on secretion of two agonists (nicotine and K+) which depend on a depolarization-dependent influx of extracellular calcium. We conclude that in addition to blocking NE uptake by inhibiting the NE transporter, the Abbott compounds may modulate peripheral NE release by inhibiting calcium flux through voltage-gated channels. This study demonstrates the utility of bovine adrenal chromaffin cells for preclinical trials of drugs that affect catecholaminergic neurotransmission.

Hormonal regulation of rat hypothalamic neuropeptide mRNAs: effect of hypophysectomy and hormone replacement on growth-hormone-releasing factor, somatostatin and the insulin-like growth factors.

Hormonal feedback regulation of hypothalamic peptides putatively involved in growth hormone (GH) regulation has been studied by measurement of steady-state mRNA levels in male hypophysectomized rats with or without thyroid hormone, corticosterone, testosterone or GH replacement. Hypothalamic GH-releasing factor (GRF) mRNA levels increased progressively following hypophysectomy to 420% of sham levels after 15 days while hypothalamic insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) mRNA levels decreased to less than 40% of sham levels. Whole hypothalamic somatostatin mRNA levels were not significantly different from sham. One week of continuous GH infusion restored hypothalamic IGF-I mRNA to levels (95%) indistinguishable from those in sham-operated controls but had no effect on either IGF-II or GRF mRNA. Thyroid hormone, corticosterone and testosterone treatment without GH had no effect on the hypophysectomy-induced reduction of either IGF-I or IGF-II mRNA levels but reversed the elevation of GRF mRNA. We conclude that hypothalamic IGF-I may be involved in GH feedback regulation and thus may function as a hypothalamic modulator of GH. In contrast, IGF-II may be regulated by one of the pituitary trophic hormones but not by GH or the target hormones tested. Finally, hypothalamic GRF mRNA regulation appears to be complex and may include target hormone feedback.

A68930: a potent and specific agonist for the D-1 dopamine receptor.

A68930 (5,6-dihydroxy-3-phenyl-1-aminomethyl-isochroman) is a potent (EC50 = 2.5 nmol/L) partial agonist at the D-1 dopamine receptor. In contrast, A68930 is a much weaker agonist (EC50 = 3,920 nmol/L) at the D-2 dopamine receptor. The orientation of the 3-phenyl substituent in the molecule is critical for the affinity and selectivity of the molecule towards the D-1 receptor in vitro and in vivo. The role of the D-1 receptor in the functioning of the basal ganglia is discussed.

Preparation of an affinity chromatography matrix for the selective purification of the dopamine D2 receptor from bovine striatal membranes.

A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.

N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity.

The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-[125I]iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-[N-acetyl-beta-glucosaminyl] asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide. Complete removal of N-linked oligosaccharide from the dopamine D2 receptor did not change the rank order potency of agonist and antagonist compounds to compete for [3H]spiperone binding to crude membrane fractions. The dopamine D2 receptor represents a highly glycosylated neural receptor.

Thyrotropin releasing hormone augments growth of spinal cord transplants in oculo.

The effects of thyrotropin releasing hormone (TRH) on spinal cord growth were evaluated using the in oculo transplant model. The growth of fetal spinal cord allografts, placed into the anterior eye chamber of Sprague-Dawley rats, was markedly augmented by acute exposure of the graft and host animal to TRH at the time of transplantation. No significant growth augmentation was seen after equimolar administration of a mixture of the amino acids that comprise the TRH molecule. It is concluded that acutely administered TRH, at the time of grafting, elicits a significant stimulation of the growth of spinal cord tissue. Our data strengthen the rationale for continued clinical trials of this peptide in spinal cord injury.

Demonstration of glucocorticoid receptor-like immunoreactivity in glucocorticoid-sensitive vasopressin and corticotropin-releasing factor neurons in the hypothalamic paraventricular nucleus.

Many parvocellular neurons in the paraventricular nucleus of the hypothalamus express high levels of corticotropin releasing factor (CRF) or vasopressin following adrenalectomy. To determine whether glucocorticoids feed back directly on these neurons, a mouse monoclonal antibody directed against the rat liver glucocorticoid receptor was used in combination with polyclonal antisera directed against either vasopressin or CRF to permit simultaneous visualization of either peptide with glucocorticoid receptor-like immunoreactivity (IR). Rats were adrenalectomized (ADX) for 2 weeks to optimize numbers of vasopressin - and CRF-IR neurons. Six hours prior to sacrifice, a separate group of adrenalectomized rats was treated with corticosterone (40 mg/kg). This short-term replacement resulted in nuclear localization of glucocorticoid receptor-like-IR but did not attenuate the increased numbers of CRF- and vasopressin-IR neurons observed after adrenalectomy. It was therefore possible to visualize vasopressin- or CFR-IR and nuclear glucocorticoid receptor-like-IR simultaneously. Cell counts of double-labeled neurons in the paraventricular nucleus of the hypothalamus (PVH) demonstrated that glucocorticoid receptor-like-IR is colocalized in virtually all the CRF and vasopressin immunoreactive parvocellular neurons studied, which respond to adrenalectomy by increased peptide expression. These data suggest that a major feedback effect of glucocorticoids on the hypothalamic-pituitary-adrenal axis is exerted directly within nuclei of CRF and vasopressin neurons.

Second messenger mechanisms governing opiate peptide transmitter regulation in the rat adrenal medulla.

Decreasing transsynaptic activity through surgical adrenal denervation or by medullary explantation, increases Leu-enkephalin immunoreactivity (Leu-Enk) and preproenkephalin mRNA (prepro-EK). Membrane depolarization prevents this rise. To determine whether depolarizing effects are mediated by intracellular movement of calcium ions, explanted medullae were depolarized in the presence of EGTA or the calcium ion 'channel' blockers D600 or verapamil. Inhibition of Ca2+ influx prevented the effects of KCl-induced depolarization on the rise in Leu-Enk and on prepro-EK. Increasing intracellular Ca2+ with the ionophore A23187, in the absence of depolarizing agents, reproduced the effects of depolarization. By contrast, medullae grown in the presence of A23187, but in Ca2+-free medium, showed similar increases in prepro-EK mRNA and Leu-Enk, indicating an absolute requirement for Ca2+. In addition, KCl-inhibitory effects could be partially blocked by the calmodulin and protein kinase-C antagonist, trifluoperazine. However, KCl effects were not antagonized by the preferential calmodulin inhibitors W7, W13 or calmidizolium even at doses 10-fold higher than required to prevent calmodulin-dependent effects. Thus, these data suggest that inhibitory effects of transsynaptic activity and membrane depolarization on adrenal enkephalin occurs through Ca2+ and perhaps through a protein kinase-C dependent pathway, mechanisms known to augment catecholamine biosynthesis. It appears then that the same or similar molecular mechanisms can result in differential regulation of these co-localized transmitter systems.

Enkephalin biosynthesis and enkephalin gene expression are increased in hippocampal mossy fibers following a unilateral lesion of the hilus.

The biosynthesis and posttranslational processing of proenkephalin and the level of preproenkephalin mRNA were investigated in the mossy fiber system of the granule cells of the hippocampus in the presence or absence of a unilateral lesion of the hilus, a procedure that produces an episode of recurrent bilateral hippocampal seizures lasting several hours. Both immunocytochemistry and radioimmunoassay (RIA) have demonstrated that the hilus lesion leads to large bilateral increases in enkephalin immunoreactivity in the mossy fiber system. In the present study, RIA data indicate that following an initial decline in immunoreactivity, enkephalin content within the mossy fibers first begins to increase between 18 and 24 hr after lesioning. Using the technique of in vivo radiolabeling and high-performance liquid chromatographic purification of identified radiolabeled peptides, we observed a 14-fold increase in incorporation of radiolabeled methionine into Met5-enkephalin at 24-30 hr postlesion, as compared with control animals, when Met5-enkephalin was purified from the mossy fiber terminal fields. To examine the posttranslational proteolytic processing of proenkephalin, the biosynthesis of 5 additional Met5-enkephalin-containing peptides was also examined. We determined that the posttranslational processing of proenkephalin did not yield exclusively penta-, hepta-, and octapeptides but larger opioid peptides as well in both control and lesioned animals, and that the ratio of the enkephalin peptides was not altered following the lesion. Measurement of preproenkephalin messenger RNA levels in the granule cells by Northern analysis revealed a marked increase following the lesion. Compared with the control animals, preproenkephalin mRNA was 8.5-fold higher in the contralateral dentate gyrus at 12 hr postlesion and 14- to 15-fold higher by 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Proenkephalin is processed in a projection-specific manner in the rat central nervous system.

The biosynthesis and posttranslational proteolytic processing of proenkephalin was studied in three projection systems in the rat central nervous system--the caudate-putamen to the globus pallidus, the paraventricular nucleus of the hypothalamus to the median eminence, and the mossy fiber system of the granule cells of the hippocampus. By using the techniques of in vivo radiolabeling and sequential high-performance liquid chromatographic purification coupled with chemical modification, the biosynthesis of six radiolabeled [Met]enkephalin-containing peptides--[Met5]enkephalin, [Met5,Arg6,Gly7,Leu8]enkephalin, [Met5,Arg6,Phe7]enkephalin, metorphamide, peptide E, and BAM 18P--was followed. In each projection system, radiolabeled enkephalins were purified to constant radiochemical specific activity. However, the posttranslational processing of proenkephalin was found to differ between these three systems, as judged by the relative ratio of these peptides. These findings imply that specific, different physiologies and behaviors may be elicited by the enkephalins based upon the specific [Met]enkephalin-containing peptides that are cleaved from proenkephalin and released in synaptic terminal fields.

Vasopressin mRNA regulation in individual hypothalamic nuclei: a northern and in situ hybridization analysis.

The present study examines the relative levels of vasopressin (AVP) mRNA within the paraventricular (PVN), supraoptic (SON), and suprachiasmatic (SCN) nuclei of the rat hypothalamus, and details the rates at which these levels change over the course of a 6 d salt-loading regimen. The quantitation of vasopressin mRNA was achieved by using three different procedures: (1) cell-free translation in rabbit reticulocyte lysate or (2) Northern analysis of poly(A)RNAs isolated from micro-punch dissected SON, PVN, and SCN, and (3) in situ hybridization histochemistry. The former involved the quantitative immunoprecipitation of the neurophysin precursors containing arginine8-vasopressin (AVP) or oxytocin, and the latter two techniques employed a radiolabeled synthetic oligodeoxynucleotide complementary to the 3' region of the AVP mRNA. Both the cell-free studies and the Northern gel analyses detected a sevenfold increase of AVP mRNA in the SON, a fivefold increase in the PVN, and no significant change in the SCN following 6 d of salt-loading. After the initiation of salt-drinking, these increases were shown to occur between 24 and 48 hr in the SON and between 48 and 72 hr in the PVN. The in situ hybridization studies revealed the anatomically correct hybridization of either 32P- or 3H-labeled AVP oligonucleotide to magnocellular perikarya within both the SON and PVN. Autoradiographic grains could be shown to be confined to the cytoplasm of these cells, and could be co-localized with immunoreactivity directed against the carboxy terminus of the AVP percursor. Comparison of x-ray level autoradiograms of control and 6 day salt-loaded SON revealed up to a sevenfold increase in specific signal in the salt-loaded sections. It is concluded that the response of AVP mRNA to osmotic stimuli in the three hypothalamic nuclei is heterogeneous, and that this heterogeneity can be explained by separating AVP neurons into two systems: one responsible for eliciting the antidiuretic actions of AVP via plasma AVP levels, and the other involved in CNS activities not directly involved with antidiuresis.

Enkephalin biosynthesis and processing during lactation.

The biosynthesis of three Met-enkephalin-containing peptides was studied in the hypothalamo-hypophysial system of normal and lactating female rats by the techniques of in vivo radiolabeling and sequential high-performance liquid chromatographic purification of radiolabeled peptides. Met-enkephalin biosynthesis was stimulated in the lactating animals; however, the products derived from the post-translational processing of the enkephalin precursor appeared to remain constant between the two states, as judged by the ratio of radioactivity present in Met5-enkephalin, Met5Arg6Gly7Leu8-enkephalin and Met5Arg6Phe7-enkephalin. Additionally, 8-fold more radioactive enkephalin was harvested from the median eminence than the posterior pituitary. Measurement of preproenkephalin mRNA in the two states also revealed an increase during lactation. All of these data suggest a physiologically important role for the enkephalins in the regulation of anterior pituitary function.

In vivo biosynthesis and transport of oxytocin, vasopressin and neurophysin from the hypothalamus to the spinal cord.

The biosynthesis of oxytocin, vasopressin and their associated neurophysins were studied in the projection from the paraventricular nucleus of the hypothalamus to the spinal cord in individual freely-moving adult male rats. Neuropeptide biosynthesis was studied in vivo by the delivery of [35S]cysteine through stereotaxically implanted indwelling cannulae using an osmotic minipump delivery system. Following the appropriate chase times, the neural lobe and spinal cord segments T1-T4 and T12-L2 were removed from fresh tissue; in addition, the nucleus of the solitary tract was punched from frozen coronal sections. The radiolabeled peptides were purified from the tissue homogenates by sequential linear and exponential gradient elution from reverse-phase high performance liquid chromatography columns. This approach has allowed us to purify radiolabeled oxytocin and vasopressin from both the upper and lower spinal cord. However, the kinetics of oxytocin and vasopressin biosynthesis appeared to be remarkably different, as judged by their differential labeling with different pulse and chase times. Additionally, the use of different chase periods following the pulse of radiolabel has allowed us to determine that oxytocin reaches the spinal cord via the fast component of axonal transport (greater than 8 mm h-1). Using immunoprecipitation and purification by high performance liquid chromatography, we were also able to purify radiolabeled neurophysins from spinal cord tissue homogenates. These results lend further support to a role for oxytocin and vasopressin in the modulation of autonomic nervous system function and to the role of the paraventricular nucleus as an integration center for endocrine and autonomic function.