Increase in CCR5 Delta32/Delta32 genotype in multiple sclerosis.

Chemokines and their receptors participate in the development of multiple sclerosis (MS) by guiding immune cells into the brain tissue. A CCR5 Delta32 deletion mutation abolishes functional CCR5 on the cell surface and may reduce cell entry into the lesion sites. To analyse the significance of this mutation in MS, we compared the frequencies of CCR5 genotype in peripheral blood mononuclear cells from 89 MS patients and 119 healthy controls. The CCR5 genotype was further compared with the CCR5 RNA and surface protein expression in 48 MS patients and their controls. In all MS patients, the Delta32/32 genotype was found with 6.7% frequency, whereas it was present only in 0.8% of the controls (6/89 vs 1/119, P = 0.01). Specifically, the Delta32/Delta32 genotype was increased (11.5%, P = 0.05) among primary progressive MS patients, whereas it was present only in 4.8% in other MS subtypes and only in 0.8% of the controls. The amount of CCR5 protein on CD4(+) cells analysed in 48 MS patients (nine primary progressive MS, 18 secondary progressive MS, 21 relapsing-remitting MS) and 13 controls decreased with genotype, being 8.9% in wt/wt, 7.7% in wt/Delta32 and 4.3% in Delta32/Delta32. CCR5 surface expression analysed on these 48 MS patients and 13 controls was significantly decreased in Delta32/Delta32 MS patients as compared with that in wt/wt genotype individuals (P = 0.004). The significantly increased number of Delta32/Delta32 individuals among our MS patients suggests that this genotype could contribute as a general risk factor for MS. However, neither the levels of RNA or surface protein correlated with MS subtype, neurological disability as expressed by expanded disability status scale, or disease progression index. Our results suggest that the lack of CCR5 does not protect from MS, but rather it may predispose to the chronic course of the disease. This would further imply that in view of the redundancy in the chemokine system, CCR5 ligands must be assumed to function through other closely related chemokine receptors.

Electrical detection of the contact between a microinjection pipette and cells.

This work presents the basic design and tests of a device designed for detecting the contact between a microinjection pipette and cell membrane. The device facilitates the automation of the microinjection procedure of living adherent cells. The measurement of the contact is based on measuring the resistance of the pipette. Breakage and clogging of the pipette can be detected with the same measurement.

The nef protein of the human immunodeficiency virus type 1 (HIV-1) inhibits a large-conductance potassium channel in human glial cells.

The Nef protein of human immunodeficiency virus type 1 (HIV-1) is abundantly expressed in HIV-1-infected astrocytes and shares amino acid sequence homology to alpha-scorpion toxins which bind to potassium channels. Here we report effects of recombinant Nef and alpha-scorpion toxins on membrane potassium transport in human U251 astroglioma cells. 86Rb+ was utilized as a tracer for potassium movements and potassium channel activity was studied by single-channel patch-clamp techniques. Results showed inactivation of a large-conductance potassium channel when Nef was added to the inner leaflet of the cell membrane of inside-out patches of U251 cells. Extracellular addition of Nef or alpha-scorpion toxins to intact glial cells did not alter potassium transport.

Serotonin induces inward potassium and calcium currents in rat cortical astrocytes.

Ca2+ imaging and patch-clamp techniques were used to study the effects of serotonin (5-HT) on ionic conductances in rat cortical astrocytes. 1 and 10 microM serotonin caused a transient increase in intracellular calcium (Ca(i)) levels in fura-2AM-loaded cultured astrocytes and in astrocytes acutely isolated and then cultured in horse serum-containing medium for over 24 h. However, the acutely isolated (less than 6 h from isolation) astrocytes, as well as acutely isolated astrocytes cultured in serum-free media, failed to respond to 5-HT by changes in Ca(i). Coinciding with the changes in Ca(i) levels, inward currents were activated by 10 microM 5-HT in cultured, but not in acutely isolated astrocytes. Two separate types of serotonin-induced, small-conductance inward single-channel currents were found. First, in both Ca2+-containing and Ca2+-free media serotonin transiently activated a small-conductance apamin-sensitive channel. Apamin is a specific blocker of the small-conductance Ca2+-activated K+ channel (sK(Ca)) When cells were pre-treated with phospholipase C inhibitor U73122 no 5-HT-induced sK(Ca) channel openings were seen, indicating that this channel was activated by Ca2+ released from intracellular stores via IP3. A second type of small inward channel activated later, but only in the presence of external Ca2+. It was inhibited by the L-type Ca2+ channel blockers, nimodipine and nifedipine. Both types of channel activity were inhibited by ketanserin, indicating activation of the 5-HT2A receptor.

Transmitter-induced calcium responses differ in astrocytes acutely isolated from rat brain and in culture.

Glial fibrillary acid protein (GFAP)-positive astrocytes isolated from the cerebral cortices of 3-10-day-old rats frequently showed increased intracellular Ca2+ concentration responses to L-glutamate and glutamate analogues. However, few of the acutely isolated cells responded to ATP, and no such cells responded to serotonin [5-hydroxytryptamine (5-HT)]. The same cell that failed to respond to ATP or 5-HT often responded to glutamate. Culturing acutely isolated cells in media containing horse serum decreased Ca2+ responses to glutamate but increased the responses to ATP and induced responses to 5-HT. In primary cultures prepared from the cerebral cortices of 1-day-old rats and cultured in horse serum, fewer of the cells responded to glutamate, but almost all cells responded to ATP and 5-HT. The lack of or limited response to, 5-HT or ATP in the acutely isolated cells seems unlikely to be due to selective damage to the respective receptors because acutely isolated GFAP-negative cells showed responses to ATP, several different proteases and mechanical dissociation yielded cells that also responded to glutamate but not to ATP, and exposure of primary cultures to papain did not abolish Ca2+ responses to several transmitters. The responses of the acutely isolated cells to glutamate but limited or lack of responses to ATP and 5-HT also correspond to what has been seen so far for astrocytes in situ. Thus, the present studies provide direct evidence that some of the receptors seen in primary astrocyte cultures may reflect a response to culture conditions and that, in the context of the relevant information so far available, acutely isolated astrocytes seem to reflect better the in vivo state.

beta-Amyloid peptide-induced morphological changes coincide with increased K+ and Cl- channel activity in rat cortical astrocytes.

Alzheimer's disease (AD) is a slowly progressing neurodegenerative disease characterized by the loss of neurons and formation of amyloid plaques, often surrounded by reactive astrocytes. Astrocytes are important regulators of the normal neuronal environment, and changed astrocyte function may lead to increased neuronal vulnerability. The slow onset of the disease with a gradual increase in the beta-amyloid peptide (beta-AP) concentrations may alter astrocyte function long before any visible symptoms of the disease are observed. We, therefore, studied in vitro the effects of small amounts of beta-AP(1-40) and -(25-35) on rat cortical astrocyte function observing changes in cell morphology, intracellular calcium levels (Cai), and ion channel activity. Incubation with 10 and 200 nM beta-APs caused increased process formation and hypertrophy. Stellation was also detected when astrocyte cultures were incubated with 1 microM AlCl3 alone, or together with beta-APs. Fura-2AM-loaded astrocytes were used to test whether the morphological changes were connected to changes in Cai levels. 1 microM beta-AP(1-40) induced transient Cai increase in approximately 17%, and beta-AP(25-35) in approximately 36% of astrocytes. In patch-clamp studies, increased K+ and Cl- channel activity was detected with 10-100 nM beta-AP(1-40). With large amounts (20 microM) of beta-AP(1-40), an additional giant channel activity emerged. These beta-AP-induced changes in astrocyte function may eventually be critical for the neuronal survival in Alzheimer's disease.

Simultaneous detection of action potential current waveforms and single ion channel openings in rat cerebellar granule cells.

Spontaneous Action Potential Current waveforms (APCs) and single ion channel openings were recorded in cultured rat cerebellar granule cells using the cell-attached configuration of the patch-clamp technique. APCs were activated without any externally applied stimuli. Both mono- and biphasic APCs were detected with a typical amplitude (positive waveform component) of 25-60 pA. Correlation between cell APCs and inward and outward single ion channel openings immediately before and after the capacitive transients was analyzed. In a detailed analysis it was found that the opening of 1.5 pA inward channel before the waveform was associated with 16%, and the opening of 1.0 pA outward channel after the waveform with 88% of APCs (of total 327 APCs analyzed). The modulation of APC activity by bath application of the amino acid taurine was found to increase the frequency of APCs, transform the APCs from mixed monophasic/biphasic to completely biphasic, and enhance the activated inward current deflections seemingly connected to the increased firing frequency.