Pipette Internal Perfusion: Methods and Applications

2007 ◽  
pp. 309-324
Author(s):  
Srinivas M. Tipparaju ◽  
Aruni Bhatnagar
Keyword(s):  
Internal Perfusion

Mechanisms of Potentiation by Calcium-Calmodulin Kinase II of Postsynaptic Sensitivity in Rat Hippocampal CA1 Neurons

1997 ◽  
Vol 78 (5) ◽  
pp. 2682-2692 ◽  
Author(s):  
Aneil M. Shirke ◽  
Roberto Malinow
Keyword(s):  
Time Course ◽  
Long Term Potentiation ◽  
Similar Process ◽  
Fluctuation Analysis ◽  
Maximal Effect ◽  
Internal Perfusion ◽  
Ca1 Neurons ◽  
Hippocampal Ca1 Neurons ◽  
Calmodulin Kinase ◽  
Hippocampal Ca1

Shirke, Aneil M. and Roberto Malinow. Mechanisms of potentiation by calcium-calmodulin kinase II of postsynaptic sensitivity in rat hippocampal CA1 neurons. J. Neurophysiol. 78: 2682–2692, 1997. Preactivated recombinant α-calcium–calmodulin dependent multifunctional protein kinase II (CaMKII*) was perfused internally into CA1 hippocampal slice neurons to test the effect on synaptic transmission and responses to exogenous application of glutamate analogues. After measurement of baseline transmission, internal perfusion of CaMKII* increased synaptic strength in rat hippocampal neurons and diminished the fraction of synaptic failures. After measurement of baseline responses to applied transmitter, CaMKII* perfusion potentiated responses to kainate but not responses to N-methyl-d-aspartate. Internal perfusion of CaMKII*potentiated the maximal effect of kainate. Potentiation byCaMKII* did not change the time course of responses to kainate, whereas increasing response size by pharmacologically manipulating desensitization or deactivation rate constants significantly altered the time course of responses. Nonstationary fluctuation analysis of responses to kainate showed a decrease in the coefficient of variation after potentiation by CaMKII*. These data support the hypothesis that CaMKII increases postsynaptic responsiveness by increasing the available number of active α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate channels and suggests that a similar process may occur during the expression of long-term potentiation.

scholarly journals Regulation of Ca2+ current in frog ventricular cardiomyocytes by guanosine 5'-triphosphate analogues and isoproterenol.

1993 ◽  
Vol 102 (3) ◽  
pp. 525-549 ◽  
Author(s):  
T D Parsons ◽  
H C Hartzell
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Ventricular Myocytes ◽  
Calcium Currents ◽  
Internal Perfusion ◽  
High Concentration ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Molecule Interactions ◽  
Gamma S

Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. To gain insight into the role of G proteins in the regulation of ICa in intact cells, the effect of internal perfusion with hydrolysis-resistant GTP analogues, guanylyl 5'-imidodiphosphate (GppNHp) or guanosine 5'-thiotriphosphate (GTP gamma S), on ICa stimulated by isoproterenol (Iso) or forskolin (Forsk) was examined. Significant differences were observed between the effects of the two GTP analogues. Internal perfusion of GppNHp resulted in a near-complete (approximately 80%) and irreversible inhibition of Iso-stimulated ICa. In contrast, internal perfusion with GTP gamma S resulted in only a partial (approximately 40%) inhibition of Iso- or Forsk-stimulated ICa. The fraction of the current not inhibited by GTP gamma S remained persistently elevated after the washout of Iso but declined to basal levels upon washout of Forsk. Excess internal GTP or GppNHp did not reduce the persistent ICa. Internal adenosine 5'-thiotriphosphate (ATP gamma S) mimicked the GTP gamma S-induced, persistent ICa. GppNHp sometimes induced a persistent ICa, but only if GppNHp was present at high concentration before Iso exposure. Inhibitors of protein kinase A inhibited both the GTP gamma S- and ATP gamma S-induced, persistent ICa. We conclude that: (a) GTP gamma S is less effective than GppNHp in inhibiting adenylyl cyclase (AC) via the inhibitory G protein, Gi; and (b) the persistent ICa results from a long-lived Gs-GTP gamma S complex that can activate AC in the absence of Iso. These results suggest that different hydrolysis-resistant nucleotide analogues may behave differently in activating G proteins and imply that the efficacy of G protein-effector molecule interactions can depend on the GTP analogue with which the G protein is activated.

Is there a second external lidocaine binding site on mammalian cardiac cells?

1989 ◽  
Vol 257 (1) ◽  
pp. H79-H84 ◽  
Author(s):  
L. A. Alpert ◽  
H. A. Fozzard ◽  
D. A. Hanck ◽  
J. C. Makielski
Keyword(s):  
Binding Site ◽  
Cardiac Arrhythmias ◽  
Local Anesthetics ◽  
Sodium Current ◽  
Cardiac Cells ◽  
Na Channel ◽  
Functional Difference ◽  
Na Channels ◽  
Internal Perfusion ◽  
Cardiac Purkinje Cells

Lidocaine and its permanently charged analogue QX-314 block sodium current (INa) in nerve, and by this mechanism, lidocaine produces local anesthesia. When administered clinically, lidocaine prevents cardiac arrhythmias. Nerve and skeletal muscle are much more sensitive to local anesthetics when the drugs are applied inside the cell, indicating that the binding site for local anesthetics is located on the inside of those Na channels. Using a large suction pipette for voltage clamp and internal perfusion of single cardiac Purkinje cells, we demonstrate that a charged lidocaine analogue blocks INa not only when applied from the inside but also from the outside, unlike noncardiac tissue. This functional difference in heart predicts that a second local anesthetic binding site exists outside or near the outside of cardiac Na channels and emphasizes that the cardiac Na channel is different from that in nerve.

scholarly journals Ion Fluxes and Short-Circuit Current in Internally Perfused Cells of Valonia ventricosa

1967 ◽  
Vol 50 (7) ◽  
pp. 1821-1834 ◽  
Author(s):  
John Gutknecht
Keyword(s):  
Marine Alga ◽  
Open Circuit Potential ◽  
Major Ions ◽  
Short Circuit ◽  
External Solution ◽  
Short Circuit Current ◽  
Open Circuit ◽  
Internal Perfusion ◽  
Ion Movements ◽  
Valonia Ventricosa

Ion transport in the giant celled marine alga, Valonia ventricosa, was studied during internal perfusion and short-circuiting of the vacuole potential. The perfusing and bathing solutions were similar to natural Valonia sap and contained the following concentrations of major ions: Na 51, K 618, and Cl 652 mM. The average short-circuit current (Isc) was 97 pEq/cm2 sec (inward positive current), and the average open-circuit potential difference (PD) was 74 mv (vacuole positive to external solution). Perfused and short-circuited cells showed a small net influx of Na (2.0 pEq/cm2 sec) and large net influxes of K (80 pEq/cm2 sec) and Cl (50 pEq/cm2 sec). Unidirectional K influx was proportional to Isc, but more than one-half of the Isc remained unaccounted for. Both the Isc and PD were partly light-dependent, declining rapidly during the first 1–2 min of darkness. Ouabain (5 x 10-4 M) had little effect on the influx of Na or K and had no effect on Iinf or PD. Fluid was absorbed at a rate of about 93 pliter/cm2 sec. Reversing the direction of fluid movement by adding mannitol to the outside solution had little effect on ion movements. The ionic and electrical properties of normal and perfused cells of Valonia are compared.

scholarly journals Effects of protein phosphatase and kinase inhibitors on the cardiac L-type Ca current suggest two sites are phosphorylated by protein kinase A and another protein kinase.

1995 ◽  
Vol 106 (3) ◽  
pp. 393-414 ◽  
Author(s):  
H C Hartzell ◽  
Y Hirayama ◽  
J Petit-Jacques
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Protein Phosphatase ◽  
Kinase Inhibitors ◽  
Delayed Rectifier ◽  
Internal Perfusion ◽  
Ca Current ◽  
Phosphatase Inhibitors ◽  
Pkc Inhibitors ◽  
Kinase A

We previously showed (Frace, A.M. and H.C. Hartzell. 1993. Journal of Physiology. 472:305-326) that internal perfusion of frog atrial myocytes with the nonselective protein phosphatase inhibitors microcystin or okadaic acid produced an increase in the L-type Ca current (ICa) and a decrease in the delayed rectifier K current (IK). We hypothesized that microcystin revealed the activity of a protein kinase (PKX) that was basally active in the cardiac myocyte that could phosphorylate the Ca and K channels or regulators of the channels. The present studies were aimed at determining the nature of PKX and its phosphorylation target. The effect of internal perfusion with microcystin on ICa or IK was not attenuated by inhibitors of protein kinase A (PKA). However, the effect of microcystin on ICa was largely blocked by the nonselective protein kinase inhibitors staurosporine (10-30 nM), K252a (250 nM), and H-7 (10 microM). Staurosporine and H-7 also decreased the stimulation of ICa by isoproterenol, but K252a was more selective and blocked the ability of microcystin to stimulate ICa without significantly reducing isoproterenol-stimulated current. Internal perfusion with selective inhibitors of protein kinase C (PKC), including the autoinhibitory pseudosubstrate PKC peptide (PKC(19-31)) and a myristoylated derivative of this peptide had no effect. External application of several PKC inhibitors had negative side effects that prevented their use as selective PKC inhibitors. Nevertheless, we conclude that PKX is not PKC. PKA and PKX phosphorylate sites with different sensitivities to the phosphatase inhibitors calyculin A and microcystin. In contrast to the results with ICa, the effect of microcystin on IK was not blocked by any of the kinase inhibitors tested, suggesting that the effect of microcystin on IK may not be mediated by a protein kinase but may be due to a direct effect of microcystin on the IK channel.

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