Back to bases
I’m gearing up to blog in a big way about a huge blind spot in classical pharmacology: chronic drug accumulation in human tissues. Protein supremacist experimental approaches have nurtured in pharmacologists an unhealthy obsession with high-affinity “drug targets,” which shrouds, and in some cases distorts, decades-old insights by physiologists into the complex mode of action of therapeutic compounds that appear structurally simple.
To warm up, I’ll present a beautiful little paper by Hiruma & Kawakami (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 2, pp. 272–279), which resuscitates dormant observations about the effects of weak bases on living cells. The authors measured the response of several essential cellular components to high concentrations of the water-soluble weak base 4-aminopyridine in primary cell cultures derived from dissected mouse dorsal root ganglia.
As shown by light microscopy (Figure 1, above), 4 mM 4-aminopyridine caused many internal compartments (vacuoles) to appear, e.g., crater-like structures in panel B, especially at the 90-minute mark. Washing away the 4-aminopyridine after a brief (minutes to hours) treatment caused all those induced vacuoles to disappear gradually. However, long-term (hours to day) treatment with 4-aminopyridine induced irreversible “vacuolization,” which was toxic to cultured neurons and non-neurons alike.
Christian de Duve, proposed a theory called lysosomotropism to describe the cellular accumulation of weak bases. It invokes the Henderson-Hasselbach equation to explain the skewed subcellular distribution of weak bases, which accumulate in acidic compartments, even synaptic vesicles.
So what, you say? 4 mM of anything you can buy from Sigma would eventually kill cells “by a non-specific mechanism.” What’s happening to other dynamic, essential cellular components besides acidic compartments during a 4-aminopyridine overdose?
Well, the authors did a few controls (Figure 3, below):
First, they verified that the 4-aminopyridine-induced vacuoles observed under differential interference contrast (DIC) are indeed lysosomes by using a fluorescent lysosomal protein marker. Next they showed that the mitochondrial and actin filament networks are basically unaffected by an acute 1-hr treatment with 4-aminopyridine (4-AP).
Then they performed a bunch of before-and-after 4-AP treatment experiments with LysoTracker Green, which “stains acidic compartments,” according to the manufacturer. I’m not exactly sure what to make of these data, other than that vacuolization is probably being conflated with the induction of autophagy.
The specificity pièce de résistance is Figure 5 (below). The V-ATPase complex, responsible for acidifying organelles, is essential for the initial vacuole induction and the ensuing cascade of physiological effects:
To be honest, parts of the discussion suffer a tad from lost in translation. Throughout the paper, I got tripped up when the authors referred to organelles within vacuoles. I assume they were talking about autophagosomes or autophagolysosomes. The fact that these words don’t roll off my tongue and I’m a native English speaker attests to the potential for confusion.
The authors concluded the paper with the following model:
“In addition, the contents of vacuoles are serous without proteins or amino acids and also without weak base. Thus, it is possible that vacuoles are formed by extrusion of H+ from acidic organelles along with water. Further studies are needed to prove this.”
I never quite understood what they meant by “serous vacuoles,” a phrase they repeated over and over. My interpretation is that they are seeing evidence of phospholipidosis. How weak base accumulation actually triggers phospholipidosis is a whole nother story…
I encourage whoever is interested to read the entire paper here. I’m hosting a discussion of it below. Who wants to break the ice?