ABSTRACT
Despite the importance of brain-mediated sympathetic activation in the morbidity and mortality of patients with high blood pressure, the precise cellular mechanisms involved remain largely unknown. We show that an imbalanced interaction between two opposing currents mediated by potassium (I(A)) and calcium (I(T)) channels occurs in sympathetic-related hypothalamic neurons in hypertensive rats. We show that this imbalance contributes to enhanced membrane excitability and firing activity in this neuronal population. Knowledge of how these opposing ion channels interact in normal and disease states increases our understanding of underlying brain mechanisms contributing to the high blood pressure condition.
Subject(s)
Calcium Channels, T-Type/physiology , Hypertension, Renovascular/physiopathology , Hypothalamus/physiopathology , Shal Potassium Channels/physiology , Sympathetic Nervous System/physiopathology , 4-Aminopyridine/pharmacology , Action Potentials/drug effects , Action Potentials/physiology , Animals , Calcium Channel Blockers/pharmacology , Calcium Channels, T-Type/genetics , Calcium Channels, T-Type/metabolism , Calcium Signaling/drug effects , Calcium Signaling/physiology , Dendrites/metabolism , Electrophysiological Phenomena/drug effects , Electrophysiological Phenomena/physiology , Gene Expression/genetics , Hypertension, Renovascular/metabolism , Hypothalamus/cytology , Male , Medulla Oblongata/cytology , Medulla Oblongata/physiology , Membrane Potentials/physiology , Neurons/drug effects , Neurons/physiology , Nickel/pharmacology , Oxytocin/metabolism , Paraventricular Hypothalamic Nucleus/cytology , Paraventricular Hypothalamic Nucleus/physiopathology , Patch-Clamp Techniques , Rats , Rats, Wistar , Shal Potassium Channels/antagonists & inhibitors , Vasopressins/metabolismABSTRACT
The hypothalamic paraventricular nucleus (PVN) is composed of functionally heterogeneous cell groups, possessing distinct electrophysiological properties depending on their functional roles. Previously, T-type Ca(2+) dependent low-threshold spikes (LTS) have been demonstrated in various PVN neuronal types, including preautonomic cells. However, the molecular composition and functional properties of the underlying T-type Ca(2+) channels have not been characterized. In the present study, we combined single cell reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry and patch-clamp recordings to identify subtypes of T-type Ca(2+) channels expressed in PVN cells displaying LTS (PVN-LTS), including identified preautonomic neurons. LTS appeared at the end of hyperpolarizing pulses either as long-lasting plateaus or as short-lasting depolarizing humps. LTS were mediated by rapidly activating and inactivating T-type Ca(2+) currents and were blocked by Ni(2+). Single cell RT-PCR and immunohistochemical studies revealed Cav3.1 (voltage-gated Ca(2+) channel) as the main channel subunit detected in PVN-LTS neurons. In conclusion, these data indicate that Cav3.1 is the major subtype of T-type Ca(2+) channel subunit that mediates T-type Ca(2+) dependent LTS in PVN neurons.
Subject(s)
Action Potentials/physiology , Calcium Channels, T-Type/metabolism , Neurons/physiology , Paraventricular Hypothalamic Nucleus/cytology , Animals , Brain Mapping , Calcium Channels, T-Type/classification , Calcium Channels, T-Type/genetics , Cholera Toxin/metabolism , Drug Interactions , Electric Stimulation/methods , In Vitro Techniques , Male , Neurons/drug effects , Neurons/radiation effects , Nickel/pharmacology , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Sodium Channel Blockers/pharmacology , Tetrodotoxin/pharmacologyABSTRACT
The complete amino-acid sequence of subunit a of the hemocyanin of the tarantula Eurypelma californicum was determined by manual sequencing. By limited chymotrypsinolysis, subunit a is split into two fragments of 25 kDa and 40 kDa, respectively, only one single peptide bond being attacked. The whole chain contains 15 methionine residues, after cyanogen bromide cleavage, 15 peptides were identified indicating that one residue (Met85) was not split by the cyanogen bromide reaction. For subcleavages, trypsin, chymotrypsin, Staphylococcus aureus proteinase, and Astacus fluviatilis proteinase were employed. The total chain length comprises 627 amino-acid residues, carbohydrate side chains were not found.
Subject(s)
Hemocyanins/chemistry , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Molecular Sequence Data , SpidersABSTRACT
The gene encoding lysostaphin of Staphylococcus staphylolyticus was cloned in Escherichia coli and its DNA sequence was determined. The complete coding region comprises 1440 base pairs corresponding to a precursor of 480 amino acids (molecular weight 51 669). It was shown by NH2-terminal amino acid sequence analysis of the purified extracellular lysostaphin from S. staphylolyticus that the mature lysostaphin consists of 246 amino acid residues (molecular weight 26926). Polyacrylamide gel electrophoresis revealed a similar molecular weight for the most active form. By computer analysis the secondary protein structure was predicted. It revealed three distinct regions in the precursor protein: a typical signal peptide (ca. 38 aa), a hydrophilic and highly ordered protein domain with 14 repetitive sequences (296 aa) and the hydrophobic mature lysostaphin. The lysostaphin precursor protein appears to be organized as a preprolysostaphin.
