Approaches for introducing peptides into intact and functional arteriolar smooth muscle: manipulation of protein kinase-based signalling.
1. An exact understanding of signal transduction pathways within intact and functional arteriolar smooth muscle is made difficult by limited access to the intracellular environment due to the cell membrane. The aim of the present studies was to determine the feasibility of using polycationic lipids and reverse permeabilization for the introduction of peptide inhibitors into smooth muscle cells of the intact arteriolar wall. 2. Isolated cannulated arterioles were exposed to polycationic lipid preparations together with varying concentrations of the protein beta-galactosidase (30-90 microg/mL). Similar experiments were also performed using cultured smooth muscle cells. Staining for the chromogenic substrate of beta-galactosidase (5-bromo-4-chloro-3-indolyl-beta-d-galactosidase; X-gal) demonstrated incorporation of the protein into cultured cells but not intact arteriolar smooth muscle. Similarly, polycationic lipid treatment did not enable loading of arteriolar smooth muscle (as assessed by cAMP-mediated vasodilation) with the protein kinase (PK) A inhibitory peptide PKI. 3. In contrast, reverse permeabilization, using high ATP concentrations in the presence of EGTA enabled introduction of PKI and inhibition of forskolin-mediated vasodilatation. Furthermore, arterioles maintained full viability following reverse permeabilization, as demonstrated by an ability to develop spontaneous myogenic tone. 4. Reverse permeabilization provides a method for introducing peptide inhibitors into functional arteriolar smooth muscle and manipulating signal transduction. Protein transfection using polycationic lipids appears to be limited by the barrier provided by the adventitia or inherent differences between cells under cultured conditions compared within the intact arteriole.
Richards
Kay
K
Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia.
Davis
Michael J
MJ
Potocnik
Simon J
SJ
Murphy
Timothy V
TV
Bishara
Nour B
NB
Rajanayagam
M A Sharmini
MA
Darby
Ian A
IA
Hill
Michael A
MA
D000818:Animals; D001160:Arterioles / Q000187:drug effects / Q000378:metabolism*; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D010446:Peptide Fragments; D010455:Peptides; D047428:Protein Kinase Inhibitors; C518603:protein kinase inhibitor peptide (5-24); D011494:Protein Kinases