Biomedical subjects

Daniel Granfeldt

Publications and source records attributed to Daniel Granfeldt.

6 recordsLinked to original sources

PubMed2006

Controlling desensitized states in ligand-receptor interaction studies with cyclic scanning patch-clamp protocols.

Ligand-gated ion channels are important control elements in regulation of cellular activities, and increasing evidence demonstrates their role as therapeutic targets. The receptors display complex desensitization kinetics, occurring on vastly different time scales. This is not only important in biology and pharmacology but might also be of technological significance since populations of receptors under microfluidic control can function analogously to DRAM memory circuits. Using a novel microfluidic method, and computer modeling of the receptor state distributions, we here demonstrate that GABAA receptor populations can be controlled to display high or low EC50 values, depending on input function (i.e., the exact pattern of agonist application). The sensitivity of the receptors can be tuned up to 40-fold (beta-alanine) by the particular agonist exposure pattern. By combining patch-clamp experiments with computer modeling of receptor state distributions, we can control the assembly of receptors in desensitized states. The technique described can be used as an analytical tool to study the effect of desensitization on the activity of ion channel effectors. We describe the differential blocking effect of the competitive antagonist bicuculline on the high- and low-EC50 GABAA receptor preparations and conclude that the inhibition is dramatically dependent on how the different desensitized states are populated. Furthermore, we show that both GABA and beta-alanine, two agonists with different affinity but similar efficacy, induce the same type of desensitization behavior and memory effects in GABAA receptors.

Animals↗

PubMed2006

A biohybrid dynamic random access memory.

We report that GABA(A) receptors in a patch-clamped biological cell form a short-term memory circuit when integrated with a scanning-probe microfluidic device. Laminar patterns of receptor activators (agonists) provided by the microfluidic device define and periodically update the data input which is read and stored by the receptors as state distributions (based on intrinsic multistate kinetics). The memory is discharged over time and lasts for seconds to minutes depending on the input function. The function of the memory can be represented by an equivalent electronic circuit with striking similarity in function to a dynamic random access memory (DRAM) used in electronic computers. Multiplexed biohybrid memories may form the basis of large-scale integrated biocomputational/sensor devices with the curious ability to use chemical signals including odorants, neurotransmitters, chemical and biological warfare agents, and many more as input signals.

Animals↗

PubMed2006

Neutrophil secretion induced by an intracellular Ca2+ rise and followed by whole-cell patch-clamp recordings occurs without any selective mobilization of different granule populations.

We have investigated calcium-induced secretion in human neutrophils, using a whole-cell patch-clamp technique. Mobilization of subcellular granules to the cell membrane was followed as the change in membrane capacitance (deltaC(m)). Both the magnitude and the kinetics of the response differed between low and high concentrations of Ca(2+). A sustained secretion following a short lag phase was induced by high concentrations of Ca(2+) (100 microM and higher). A stable plateau was reached after 5-7 minutes at deltaC(m) values corresponding to values expected after all specific as well as azurophil granules have been mobilized. Capacitance values of the same magnitude could be obtained also at lower Ca(2+) concentrations, but typically no stable plateau was reached within the measuring time. In contrast to previous studies, we were unable to detect any pattern of secretion corresponding to a distinct submaximal response or selective mobilization of granule subsets specified by their Ca(2+)-sensitivity.

Journal Article↗

PubMed2005

A chemical waveform synthesizer.

Algorithms and methods were developed to synthesize complex chemical waveforms in open volumes by using a scanning-probe microfluidic platform. Time-dependent variations and oscillations of one or several chemical species around the scanning probe, such as formation of sine waves, damped oscillations, and generation of more complex patterns, are demonstrated. Furthermore, we show that intricate bursting and chaotic calcium oscillations found in biological microdomains can be reproduced and that a biological cell can be used as a probe to study receptor functionalities as a function of exposure to time-dependent variations of receptor activators and inhibitors. Thus, the method allows for studies of biologically important oscillatory reactions. More generally, the system allows for detailed studies of complex time-varying chemical and physical phenomena in solution or at solution/surface interfaces.

Algorithms↗

PubMed2003

Reactivation of formyl peptide receptors triggers the neutrophil NADPH-oxidase but not a transient rise in intracellular calcium.

In neutrophils, coupling of chemoattractants to their cell surface receptor at low temperature (<or=15 degrees C) leads to receptor deactivation/desensitization without any triggering of the superoxide anion-generating NADPH-oxidase. We show that the deactivated formyl peptide receptors (FPRs) can be reactivated/resensitized by the cytoskeleton-disrupting drug cytochalasin B. Such cytoskeleton-dependent receptor reactivation occurs also with the closely related receptors FPR-like-1 and C5aR but not with the receptors for interleukin-8 and platelet-activating factor. The reactivation state was further characterized with FPR as a model. The signals generated by receptor reactivation induced superoxide production that was terminated in 5-8 min, after which the neutrophils entered a new state of homologous deactivation. FPR antagonists were potent inhibitors of the superoxide production induced by the reactivated receptors, suggesting that the occupied receptors turn into an actively signaling state when the cytoskeleton is disrupted. The signals generated by the reactivated receptor were pertussis toxin-sensitive, indicating involvement of a G-protein. However, no transient elevation of intracellular Ca2+ accompanies the NADPH-oxidase activation. This was not due to a general down-regulation of phospholipase C/Ca2+ signaling, and despite the fact that no intracellular Ca2+ transient was generated, protein kinase C still appeared to be involved in the response. Further, phosphatidylinositol 3-kinase, mitogen-activated protein kinase, and MEK all participated in the generation of second messengers from the reactivated receptors.

Androstadienes↗

PubMed2002

Capacitative Ca2+ influx and activation of the neutrophil respiratory burst. Different regulation of plasma membrane- and granule-localized NADPH-oxidase.

The neutrophil NADPH-oxidase may be activated in the plasma membrane, resulting in release of oxygen metabolites extracellularly, or in the granule or phagosomal membranes, giving intracellular production of oxidants. An increase in [Ca2+]i mediated through binding of fMLF to its receptor is part of a signaling cascade that activates the plasma membrane-localized oxidase. In contrast, a rise in [Ca2+]i induced by a Ca2+ ionophore results in activation of the intracellular pool of oxidase. We mimicked fMLF-induced emptying of intracellular Ca2+ stores with thapsigargin. This induced a pronounced intracellular oxidase activity but no extracellular release of oxidants. The thapsigargin-induced effect was dependent on capacitative Ca2+ influx, because the effect was inhibited dose-dependently by EGTA and the Ca2+ channel blocker La3+. At La3+ concentrations between 200 and 400 microM, thapsigargin also induced a massive extracellular production of superoxide anion. No other channel blockers tested induced a similar effect. We conclude that elevation in [Ca2+]i by capacitative Ca2+ influx induces NADPH-oxidase activation at an intracellular site. Further, activation of the plasma membrane-localized NADPH-oxidase is regulated by a more complex Ca2+ signaling, involving capacitative Ca2+ influx and possibly the specific action of La3+-sensitive Ca2+ channels.

Calcium↗